Crystalline forms of eravacycline

ABSTRACT

The invention relates to crystalline forms of the bis-HCl salt of a compound represented by Structural Formula 1, and pharmaceutical compositions comprising crystalline forms of the bis-HCL salt of a compound represented by Structural Formula 1 described herein. The crystalline forms of the bis-HCl salt of a compound of Structural Formula 1 and compositions comprising the crystalline forms of the compound of Structural Formula 1 provided herein, in particular, crystalline Form I, crystalline Form J, crystalline Form A, and crystalline Form B, or mixtures thereof, can be incorporated into pharmaceutical compositions, which can be used to treat various disorders. Also described herein are methods for preparing the crystalline forms (e.g., Forms I, J, B and A) of the bis-HCl salt of a compound represented by Structural Formula 1.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/342,779, filed on Apr. 17, 2019, which is the U.S. National Stage ofInternational Application No. PCT/US2017/57385, filed on Oct. 19, 2017,published in English, which claims the benefit of U.S. ProvisionalApplication No. 62/410,230, filed on Oct. 19, 2016. The entire teachingsof the above application is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Contract No.HHSO100201200002C and Subcontract No. 7834S1 awarded by the Departmentof Health and Human Services. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

The tetracyclines are broad spectrum anti-microbial agents that arewidely used in human and veterinary medicine. The total production oftetracyclines by fermentation or semi-synthesis is measured in thethousands of metric tons per year.

The widespread use of tetracyclines for therapeutic purposes has led tothe emergence of resistance to these antibiotics, even among highlysusceptible bacterial species. Tetracycline analogs having improvedantibacterial activities and efficacies against other tetracyclineresponsive diseases or disorders have been described (see, for example,U.S. Pat. No. 8,796,245). A particularly potent compound is eravacycline(7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline), whichhas the chemical structure shown in Structural Formula 1:

The solid form of a compound can be important in the formulation ofpharmaceutical compositions. For example, crystalline and amorphousforms of a compound can have different physical properties (e.g.,stability, dissolution rate, density, etc.) relating to theirsuitability for use in pharmaceutical compositions. The difference inphysical properties can also affect a crystalline or amorphous form'susefulness, for example, as an intermediate in the synthesis of a formsuitable for use in pharmaceutical compositions.

There is a need for crystalline forms of eravacycline that arethermodynamically stable and suitable for use in pharmaceuticalcompositions (e.g., are readily dissolvable, exhibit good flowproperties and/or good chemical stability). There is a further need forcrystalline forms of eravacycline having physical properties that enablethe manufacture of eravacycline for use in pharmaceutical compositionsin high yield and high purity.

SUMMARY OF THE INVENTION

The present invention relates to crystalline forms of the bis HCl saltof the compound represented by Structural Formula 1, designatedcrystalline Form A, crystalline Form B, crystalline Form I andcrystalline Form J and compositions comprising the crystalline forms.

In one embodiment, a crystalline form of a compound represented by thebis HCl salt of Structural Formula 1 is provided, wherein thecrystalline form is Form I. In this embodiment, crystalline Form I ischaracterized by at least three x-ray powder diffraction peaks at 2θangles selected from 7.22°, 7.80°, 10.41°, and 11.11°.

In another embodiment, a crystalline form of a compound represented bybis HCl salt of Structural Formula 1 is provided, wherein thecrystalline form is Form J. In this embodiment, crystalline Form J ischaracterized by at least three x-ray powder diffraction peaks at 2θangles selected from 7.02°, 7.80°, 22.13°, and 23.22°.

In yet another embodiment, a crystalline form of a compound representedby the bis HCl salt of Structural Formula 1 is provided, wherein thecrystalline form is Form A. In this embodiment, crystalline Form A ischaracterized by at least three x-ray powder diffraction peaks at 2θangles selected from 3.31°, 6.01°, 6.33°, and 8.73°.

In another embodiment, a crystalline form of a compound represented bythe bis HCl salt of Structural Formula 1 is provided, wherein thecrystalline form is Form B. In this embodiment, crystalline Form B ischaracterized by at least three x-ray powder diffraction peaks at 2θangles selected from 9.19°, 9.66°, 23.32°, and 24.35°.

Another embodiment, is a composition comprising particles of one or morecrystalline forms of a compound represented by the bis HCl salt ofStructural Formula 1:

wherein the particles are selected from: particles of crystalline Form Icharacterized by at least three x-ray powder diffraction peaks at 2θangles selected from 7.22°, 7.80°, 10.41°, and 11.11°; particles ofcrystalline form Form J characterized by at least three x-ray powderdiffraction peaks at 2θ angles selected from 7.02°, 7.80°, 22.13°, and23.22°; particles of crystalline Form A characterized by at least threex-ray powder diffraction peaks at 2θ angles selected from 3.31°, 6.01°,6.33°, and 8.73°; and particles of crystalline Form B characterized byat least three x-ray powder diffraction peaks at 2θ angles selected from9.19°, 9.66°, 23.32°, and 24.35°. In a specific aspect, the compositioncomprises particles of crystalline Form I and crystalline form J. Inanother aspect, the weight percent of crystalline Form J in thecomposition is 25% or less (e.g., about 20% or less, about 15% or less,about 10% or less, about 5% or less or about 1%).

Another embodiment is a pharmaceutical composition comprising particlesof Form I, Form J, Form A, Form B or mixtures thereof and apharmaceutically acceptable carrier.

A further embodiment is a method for treating or preventing atetracycline-responsive disease or disorder, the method comprisingadministering to a subject in need thereof a therapeutically orprophylactically effective amount of crystalline Form I, Form J, Form A,Form B or a mixture thereof. In one aspect, the tetracycline-responsivedisease or disorder is an infection. In a specific aspect, the infectionis caused by bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention.

FIG. 1 is an x-ray powder diffraction (XRPD) pattern of StructuralFormula 1 as the bis HCl salt)—Form A.

FIG. 2A is a thermogravimetric analysis (TGA) thermogram of StructuralFormula 1—Form A.

FIG. 2B is a differential scanning calorimetry (DSC) thermogram ofStructural Formula 1—Form A.

FIG. 3 is an XRPD pattern of Structural Formula 1—Form B.

FIG. 4A is a TGA thermogram of Structural Formula 1—Form B.

FIG. 4B is a DSC thermogram of Structural Formula 1—Form B.

FIG. 5 is an XRPD pattern of Structural Formula 1—Form I.

FIG. 6A is a TGA thermogram of Structural Formula 1—Form I.

FIG. 6B is a DSC thermogram of Structural Formula 1—Form I

FIG. 7 is an XRPD pattern of Structural Formula 1—Form J.

FIG. 8A is a TGA thermogram of Structural Formula 1—Form J.

FIG. 8B is a DSC thermogram of Structural Formula 1—Form J.

FIG. 9 is a DVS of Structural Formula 1—Amorphous Form

FIG. 10 is a DVS of Structural Formula 1—Form A

FIG. 11 is a DVS of Structural Formula 1—Form B

FIG. 12 is a DVS of Structural Formula 1—Form I

FIG. 13 is a DVS of Structural Form 1—Form J

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Crystalline Forms of Eravacycline

Provided herein are crystalline forms of the bis HCl salt of thecompound represented by Structural Formula 1, designated crystallineForm A, crystalline Form B, crystalline Form I and crystalline Form J.

“Crystalline,” as used herein, refers to a homogeneous solid formed by arepeating, three-dimensional pattern of atoms, ions or molecules (e.g.,an anhydrous molecule or a salt thereof, solvate thereof, or combinationof the foregoing) having fixed distances between constituent parts. Theunit cell is the simplest repeating unit in this pattern.

A crystalline form provided herein can be a single crystalline form orcan comprise a mixture of two or more different crystalline forms. Forexample, in some embodiments, crystalline Forms A, B, I, and J of acompound of Structural Formula 1 are provided as single crystallineforms (i.e., single crystalline Form A, single crystalline Form B,single crystalline Form I, single crystalline Form J). Alternatively, acrystalline form can comprise a mixture of two or more crystalline formsof a compound of Structural Formula 1 (e.g., a mixture of two or more ofcrystalline Forms A, B, I, and J, specifically, of crystalline Forms Iand J).

“Single crystalline form,” as used herein, refers to a single crystal ofa crystalline solid or a plurality of crystals of a crystalline solidwherein each of the plurality of crystals has the same crystal form.

The crystalline forms provided herein can be identified on the basis ofcharacteristic peaks in an x-ray powder diffraction (XRPD) analysis.XRPD is a scientific technique that measures the x-rays, neutrons orelectrons scattered by a powder or microcrystalline material as afunction of scattering angle. XRPD can be used to identify andcharacterize crystalline solids, as the diffraction pattern produced bya particular solid is typically distinctive to that solid and can beused as a “fingerprint” to identify that solid. For example, an XRPDpattern or diffractogram (e.g., a pattern or diffractogram produced by asample, such as an unknown sample) that is substantially in accordancewith a reference XRPD pattern or diffractogram can be used to determinethe identity between the sample material and the reference material.Both the position and the relative intensity of the peaks in an XRPDdiffractogram are indicative of the particular phase and identity of amaterial.

FIGS. 1, 3, 5, and 7 show XRPD patterns of various crystalline formsdescribed herein. An XRPD pattern that is “substantially in accordance”with one or more figures herein showing an XRPD pattern or diffractogramis an XRPD pattern that would be considered by one skilled in the art torepresent the same crystalline form of the compound of StructuralFormula 1 as the sample of the compound of Structural Formula 1 thatprovided the XRPD pattern of one or more figures provided herein. Thus,an XRPD pattern that is substantially in accordance may be identical tothat of one of the figures or, more likely, may be somewhat differentfrom one or more of the figures. An XRPD pattern that is somewhatdifferent from one or more of the figures may not necessarily show eachof the lines of the diffraction pattern presented herein and/or may showa slight change in appearance or intensity of the lines or a shift inthe position of the lines. These differences typically result fromdifferences in the conditions involved in obtaining the data ordifferences in the purity of the sample used to obtain the data. Aperson skilled in the art is capable of determining if a sample of acrystalline compound is of the same form as or a different form from aform disclosed herein by comparison of the XRPD pattern of the sampleand the corresponding XRPD pattern disclosed herein.

It is to be understood that any 2θ angle specified herein means thespecified value±0.2°. For example, when a described embodiment or aclaim specifies a 2θ of 4.4°, this is to be understood to mean4.4°±0.2°, that is, a 2θ angle of from 4.2° to 4.6°.

The crystalline forms provided herein can also be identified on thebasis of differential scanning calorimetry (DSC) and/orthermogravimetric analysis (TGA). DSC is a thermoanalytical technique inwhich the difference in the amount of heat required to increase thetemperature of a sample is measured as a function of temperature. DSCcan be used to detect physical transformations, such as phasetransitions, of a sample. For example, DSC can be used to detect thetemperature(s) at which a sample undergoes crystallization, melting orglass transition.

TGA is a method of thermal gravimetric analysis in which changes inphysical and chemical properties of a material are measured as afunction of increasing temperature (with constant heating rate) or as afunction of time (with constant temperature and/or constant mass loss).TGA can provide information about physical phenomena, such assecond-order phase transitions, or about chemical phenomena, such asdesolvation and/or decomposition.

FIGS. 2B, 4B, 6B and 8B show DSC thermograms of various crystallineforms described herein. FIGS. 2A, 4A, 6A, and 8A show TGA thermograms ofvarious crystalline forms described herein. A DSC or TGA thermogram thatis “substantially in accordance” with one or more figures herein showinga DSC or TGA thermogram is a DSC or TGA thermogram that would beconsidered by one skilled in the art to represent the same crystallineform of the compound of Structural Formula 1 as the sample of thecompound of Structural Formula 1 that provided the DSC or TGA thermogramof one or more figures provided herein.

It is to be understood that any temperature associated with DSC or TGAspecified herein means the specified value±5° C. or less. For example,when an embodiment or a claim specifies an endothermic peak at about179° C., this is to be understood to mean 179° C.±5° C. or less, that isa temperature of from 174° C. to 184° C. In preferred embodiments, a DSCor TGA temperature is the specified value±3° C., in more preferredembodiments, ±2° C.

In some embodiments, crystalline forms are solvates. “Solvate,” as usedherein, refers to a chemical compound formed by the interaction of asolute (e.g., a compound of Structural Formula 1) and one or moresolvents (e.g., methanol, ethanol, water). Thus, “solvate” includessolvates containing a single type of solvent molecule and solvatescontaining more than one type of solvent molecule (mixed solvates orco-solvates). Typically, the one or more solvents in solvates describedherein is an organic solvent or a combination of organic solvents,although water can also form solvates, called hydrates.

For example, Form I described herein has been characterized as ahemi-ethanolate with 0.5 eq. of ethanol. Form J described herein hasbeen characterized as the desolvated Form of I.

Form I:

In a first embodiment, a crystalline form of a compound represented bythe bis HCl salt of Structural Formula 1 is provided, wherein thecrystalline form is Form I and is characterized by at least three x-raypowder diffraction peaks at 2θ angles selected from 7.22°, 7.80°,10.41°, and 11.11°, or at least four x-ray powder diffraction peaks at2θ angles selected from 7.22°, 7.80°, 8.19°, 10.41°, and 11.11°, atleast five x-ray powder diffraction peaks at 2θ angles selected from7.22°, 7.80°, 8.19°, 10.41°, 11.11°, 15.00°, 16.47° and 20.44°. In aparticular embodiment, Form I is characterized by x-ray powderdiffraction peaks at 2θ angles of 7.22°, 7.80°, 10.41°, and 11.11°, orby x-ray powder diffraction peaks at 2θ angles of 7.22°, 7.80°, 8.19°,10.41°, and 11.11°, or by x-ray powder diffraction peaks at 2θ angles of7.22°, 7.80°, 8.19°, 10.41°, 11.11°, 15.00°, 16.47° and 20.44° or byx-ray powder diffraction peaks at 2θ angles of 7.22°, 7.80°, 8.19°,10.41°, 11.11°, 12.17°, 13.52°, 15.00°, 15.70°, 16.47°, 19.96°, and20.44°. In some embodiments, crystalline Form I is characterized by anx-ray powder diffraction pattern substantially in accordance with thatdepicted in FIG. 5.

Crystalline Form I may be further characterized by a differentialscanning calorimetry thermogram comprising a broad, weak endothermicpeak at about 116° C. or a weak endothermic peak at about 171° C. Insome embodiments, the TGA thermogram and/or the DSC thermogram aresubstantially in accordance with those in FIG. 6A or FIG. 6B.

In a particular embodiment, Form I is in the form of a solvate, forexample, an ethanol solvate, such as a hemi-ethanolate comprising about0.5 molar equivalents of solute per molar equivalent of the compound ofStructural Formula 1.

In another embodiment, Form I is in the form of a co-solvate. In aparticular embodiment, the co-solvate is a dihydrate and hemi-ethanolco-solvate.

Form J:

In a second embodiment, a crystalline form of a compound represented bybis HCl salt of Structural Formula 1 is provided, wherein thecrystalline form is Form J and is characterized by at least three x-raypowder diffraction peaks at 2θ angles selected from 7.02°, 7.80°,22.13°, and 23.22°, at least four x-ray powder diffraction peaks at 2θangles selected from 7.02°, 7.80°, 10.25°, 22.13°, and 23.22°, or atleast five x-ray powder diffraction peaks at 2θ angles selected from7.02°, 7.80°, 10.25°, 11.00°, 13.29°, 13.60°, 14.98°, 21.92°, 22.13°,23.22°, 24.02° and 25.28°.

In a particular embodiment, Form J is characterized by x-ray powderdiffraction peaks at 2θ angles of 7.02°, 7.80°, 22.13°, and 23.22°, orby x-ray powder diffraction peaks at 2θ angles of 7.02°, 7.80°, 10.25°,22.13°, and 23.22° or by x-ray powder diffraction peaks at 2θ angles of7.02°, 7.80°, 10.25°, 11.00°, 13.29°, 13.60°, 14.98°, 21.92°, 22.13°,23.22°, 24.02° and 25.28°. In another particular embodiment, Form J ischaracterized by x-ray powder diffraction peaks at 2θ angles of 7.02°,7.80°, 10.25°, 11.00°, 11.85°, 13.29°, 13.60°, 14.98°, 15.27°, 16.21°,16.39°, 17.04°, 20.10°, 21.53°, 21.92°, 22.13°, 22.52°, 23.22°, 24.02°,24.41°, 25.28°, 26.08°, 26.35°, 26.78°, and 27.90°. In some embodiments,crystalline Form J is characterized by an x-ray powder diffractionpattern substantially in accordance with that depicted in FIG. 7.

Crystalline Form J may be further characterized by a differentialscanning calorimetry thermogram comprising a broad endothermic peak at118° C. In some embodiments, the TGA thermogram and/or the DSCthermogram are substantially in accordance with those in FIG. 8A or FIG.8B.

Form A:

In a third embodiment, a crystalline form of a compound represented bythe bis HCl salt of Structural Formula 1 is provided, wherein thecrystalline form is Form A and is characterized by at least three x-raypowder diffraction peaks at 2θ angles selected from 3.31°, 6.01°, 6.33°,and 8.73°, at least four x-ray powder diffraction peaks at 2θ anglesselected from 3.31°, 6.01°, 6.33°, 8.73°, and 14.06°, or at least fivex-ray powder diffraction peaks at 2θ angles selected from 3.31°, 6.01°,6.33°, 8.73°, 10.49°, 14.06° and 16.55°. In a particular embodiment,Form A is characterized by x-ray powder diffraction peaks at 2θ anglesof 3.31°, 6.01°, 6.33°, and 8.73°, or by x-ray powder diffraction peaksat 2θ angles of 3.31°, 6.01°, 6.33°, 8.73°, and 14.06°, or by x-raypowder diffraction peaks at 2θ angles of 3.31°, 6.01°, 6.33°, 8.73°,10.49°, 14.06° and 16.55°, In some embodiments, crystalline Form A ischaracterized by an x-ray powder diffraction pattern substantially inaccordance with that depicted in FIG. 1.

Crystalline Form A may be further characterized by a differentialscanning calorimetry thermogram comprising a broad, endothermic peak atabout 120° C. In some embodiments, the TGA thermogram and/or the DSCthermogram are substantially in accordance with those in FIG. 2A or FIG.2B.

In a particular embodiment, Form A is in the form of a solvate or aco-solvate, for example, a water and ethanol co-solvate. In a particularembodiment, the water and ethanol co-solvate is a variable co-solvate.

Form B:

In a fourth embodiment, a crystalline form of a compound represented bythe bis HCl salt Structural Formula 1 is provided, wherein thecrystalline form is Form B and is characterized by at least three x-raypowder diffraction peaks at 2θ angles selected from 9.19°, 9.66°,23.32°, and 24.35°, by at least four x-ray powder diffraction peaks at2θ angles selected from 9.19°, 9.66°, 17.63°, 23.32°, and 24.35°, or byat least five x-ray powder diffraction peaks at 2θ angles selected from6.10°, 9.19°, 9.48°, 9.66°, 13.05°, 17.63°, 17.77°, 19.94°, 20.48°,23.32° and 24.35°. In a particular embodiment, Form B is characterizedby x-ray powder diffraction peaks at 2θ angles of 9.19°, 9.66°, 23.32°,and 24.35°, or by x-ray powder diffraction peaks at 2θ angles of 9.19°,9.66°, 17.63°, 23.32°, and 24.35°, or by x-ray powder diffraction peaksat 2θ angles of 6.10°, 9.19°, 9.48°, 9.66°, 13.05°, 17.63°, 17.77°,19.94°, 20.48°, 23.32°, 23.87°, and 24.35°, or by x-ray powderdiffraction peaks at 2θ angles of 6.10°, 9.19°, 9.48°, 9.66°, 12.08°,13.05°, 17.63°, 17.77°, 19.54°, 19.94°, 20.48°, 23.32°, 23.87°, and24.35°. In some embodiments, crystalline Form B is characterized by anx-ray powder diffraction pattern substantially in accordance with thatdepicted in any of FIG. 3.

Crystalline Form B may be further characterized by a differentialscanning calorimetry thermogram of FIG. 4B and a melting peak of 128° C.In some embodiments, the TGA thermogram and/or the DSC thermogram aresubstantially in accordance with those in FIG. 4A or FIG. 4B.

In a particular embodiment, Form B is in the form of a solvate or aco-solvate, for example, a water and methanol co-solvate.

Crystalline Form B can be prepared, in accordance with the methodsdisclosed herein, at a desirable purity and yield. The exceptionalpurity of crystalline Form B can be translated into highly purecrystalline Form I, crystalline Form J or a mixture of crystalline FormsI and J for pharmaceutical use. By using the procedures described hereinfor converting crystalline Form B into crystalline Forms I, crystallineForm J or a mixture thereof, crystalline Form I, crystalline Form J or amixture thereof can be isolated as a composition ready for formulationas a pharmaceutical composition (e.g., acceptable purity, readilydissolvable and/or exhibiting good flow properties).

Compositions and Pharmaceutical Compositions

In another embodiment, the invention relates to a composition,comprising particles of one or more crystalline forms of a compoundrepresented by the bis HCl salt of Structural Formula 1:

-   -   wherein, the one or more crystalline forms are selected from:        -   crystalline Form I characterized by at least three x-ray            powder diffraction peaks at 2θ angles selected from 7.22°,            7.80°, 10.41°, and 11.11′;    -   crystalline form Form J characterized by at least three x-ray        powder diffraction peaks at 2θ angles selected from 7.02°,        7.80°, 22.13°, and 23.22°;    -   crystalline Form A characterized by at least three x-ray powder        diffraction peaks at 2θ angles selected from 3.31°, 6.01°,        6.33°, and 8.73°; and    -   crystalline Form B characterized by at least three x-ray powder        diffraction peaks at 2θ angles selected from 9.19°, 9.66°,        23.32°, and 24.35°.

In a particular embodiment, the composition comprises particles that area mixture of crystalline Form I and crystalline Form J. In one aspect,the weight percent of crystalline Form J in the composition is 25% orless (e.g., about 20% or less, about 15% or less, about 10% or less,about 5% or less or about 1%.

Also provided herein are pharmaceutical compositions comprising acrystalline form (e.g., Form A, B, I, or J) of compositions describedherein and a pharmaceutically acceptable carrier. The compositioncomprises particles of one or more crystalline form (e.g., Form A, B, I,or J) of the bis HCl salt of a compound of Structural Formula 1. Forexample, the pharmaceutical composition comprises Form I, Form J, FormA, Form B or a combination of more than one of Form I, Form J, Form Aand Form B. In a particular embodiment, the pharmaceutical compositioncomprises a mixture of Form I and Form J.

In certain embodiments, the invention relates to the compositions andpharmaceutical compositions as described herein, wherein the crystallineform is characterized by at least three x-ray powder diffraction peaksat 2θ angles selected from 7.22°, 7.80°, 10.41°, and 11.11°, at leastfour x-ray powder diffraction peaks at 2θ angles selected from 7.22°,7.80°, 8.19°, 10.41°, and 11.11°, or at least five x-ray powderdiffraction peaks at 2θ angles selected from 7.22°, 7.80°, 8.19°,10.41°, 11.11°, 15.00°, 16.47° and 20.44°.

In certain embodiments, the invention relates to compositions andpharmaceutical compositions described herein, wherein the crystallineform is characterized by x-ray powder diffraction peaks at 2θ angles of7.22°, 7.80°, 10.41°, and 11.11°, by x-ray powder diffraction peaks at2θ angles of 7.22°, 7.80°, 8.19°, 10.41°, and 11.11°, or by x-ray powderdiffraction peaks at 2θ angles of 7.22°, 7.80°, 8.19°, 10.41°, 11.11°,15.00°, 16.47° and 20.44°.

In certain embodiments, the invention relates to compositions andpharmaceutical compositions described herein, wherein the crystallineform is characterized by an x-ray powder diffraction patternsubstantially in accordance with that depicted in FIG. 5.

In certain embodiments, the invention relates to compositions andpharmaceutical compositions described herein, wherein the crystallineform is further characterized by a differential scanning calorimetrythermogram comprising a broad, weak endothermic peak at about 116° C. ora weak endothermic peak at about 171° C. In certain embodiments, theinvention relates to any of the compositions described herein, whereinthe crystalline form is further characterized by a differential scanningcalorimetry thermogram comprising a broad, weak endothermic peak atabout 116° C. In certain embodiments, the invention relates to any ofthe compositions described herein, wherein the crystalline form isfurther characterized by a differential scanning calorimetry thermogramcomprising a weak endothermic peak at about 171° C.

In certain embodiments, the invention relates to the compositions andpharmaceutical compositions as described herein, wherein the crystallineform is characterized by at least three x-ray powder diffraction peaksat 2θ angles selected from 7.02°, 7.80°, 22.13°, and 23.22°, at leastfour x-ray powder diffraction peaks at 2θ angles selected from 7.02°,7.80°, 10.25°, 22.13°, and 23.22°, or at least five x-ray powderdiffraction peaks at 2θ angles selected from 7.02°, 7.80°, 10.25°,11.00°, 13.29°, 13.60°, 14.98°, 21.92°, 22.13°, 23.22°, 24.02°, 25.28°.

In certain embodiments, the invention relates to any of the compositionsdescribed herein, wherein the crystalline form is characterized by x-raypowder diffraction peaks at 2θ angles of 7.02°, 7.80°, 22.13°, and23.22°, or by x-ray powder diffraction peaks at 2θ angles of 7.02°,7.80°, 10.25°, 22.13°, and 23.22, or by x-ray powder diffraction peaksat 2θ angles of 7.02°, 7.80°, 10.25°, 11.00°, 13.29°, 13.60°, 14.98°,21.92°, 22.13°, 23.22°, 24.02° and 25.28° or by x-ray powder diffractionpeaks at 2θ angles of 7.02°, 7.80°, 10.25°, 11.00°, 11.85°, 13.29°,13.60°, 14.98°, 15.27°, 16.21°, 16.39°, 17.04°, 20.10°, 21.53°, 21.92°,22.13°, 22.52°, 23.22°, 24.02°, 24.41°, 25.28°, 26.08°, 26.35°, 26.78°,and 27.90°.

In certain embodiments, the invention relates to compositions orpharmaceutical compositions described herein, wherein the crystallineform is characterized by an x-ray powder diffraction patternsubstantially in accordance with that depicted in FIG. 7.

In certain embodiments, the invention relates to any of the compositionsdescribed herein, wherein the crystalline form is further characterizedby a differential scanning calorimetry thermogram comprising a broadendothermic peak at 118° C.

In certain embodiments, the invention relates to any of the compositionsdescribed herein, wherein the crystalline form is characterized by anx-ray powder diffraction pattern having a widened shoulder on the peakat 7.2° 20 as compared to the x-ray powder diffraction pattern ofparticles of Form I.

In certain embodiments, the invention relates to any of the compositionsdescribed herein, wherein the composition comprises a mixture ofparticles of Form I and particles of Form J; and the crystalline form ischaracterized by an x-ray powder diffraction pattern having a widenedshoulder on the peak at 7.2° 20 as compared to the x-ray powderdiffraction pattern of particles of Form I.

The term “pharmaceutically acceptable carrier” means a non-toxicsolvent, dispersant, excipient, adjuvant or other material which ismixed with the active ingredient in order to permit formation of apharmaceutical composition, i.e., a dosage form capable of beingadministered to a subject. A “pharmaceutically acceptable carrier”should not destroy the activity of the compound with which it isformulated. Pharmaceutically acceptable carriers are well known in theart.

Pharmaceutically acceptable carriers, adjuvants or vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

Pharmaceutical compositions of the invention may be administered orally,parenterally (including subcutaneous, intramuscular, intravenous andintradermal), by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. In some embodiments,provided pharmaceutical compositions are administrable intravenouslyand/or intraperitoneally.

The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intraocular, intravitreal, intra-articular,intra-synovial, intrasternal, intrathecal, intrahepatic, intraperitonealintralesional and intracranial injection or infusion techniques.Preferably, the pharmaceutical compositions are administered orally,subcutaneously, intraperitoneally or intravenously. Sterile injectableforms of the pharmaceutical compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium.

Pharmaceutical compositions of this invention may be orally administeredin any orally acceptable dosage form including, but not limited to,capsules, tablets, aqueous suspensions or solutions. In the case oftablets for oral use, carriers commonly used include lactose and cornstarch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added. In some embodiments, aprovided oral formulation is formulated for immediate release orsustained/delayed release. In some embodiments, the composition issuitable for buccal or sublingual administration, including tablets,lozenges and pastilles. A provided compound can also be inmicro-encapsulated form.

Specific pharmaceutically acceptable carriers suitable for use in anoral formulation such as a tablet or capsule include, but are notlimited to, microcrystalline cellulose (Avicel PH101), croscarmelloseSodium (Ac-Di-Sol), kollidon 30 powder (polyvinylpyrrolidone, povidone),colloidal silicon dioxide M5-P, magnesium stearate, microcrystallinecellulose (Avcel PH102), sodium lauryl sulfate (Kolliphor SLS Fine) andColloidal Silicon Dioxide M5-P. Each of the above listed carriers can beused in an oral formulation either alone or in any combination.

Further pharmaceutically acceptable carriers suitable for use in an oralformulation such as a tablet or capsule include, but are not limited to,microcrystalline cellulose (Avicel PH112), crospovidone (polyplasdoneXL-10), colloidal silicone dioxide (Cab-O-Sil M-5P), Talc, starch andcalcium stearate. In a particular aspect, the crystalline form (e.g.,Form A, Form B, Form I, Form J or a mixture of Form I and Form J) ispresent in the oral formulation from about 25-45% by weight (freebaseweight). In other aspects, Disodium EDTA is also present in the oralformulation. In certain aspects, the EDTA increases the bioavailabilityof the active. In a particular embodiment, the bioavailability of theactive is increased by from about 1.5 fold to about 20 fold (e.g., 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20fold). When EDTA is present in the formulation, the w/w ratio of thecrystalline form (freebase weight) to EDTA ranges from about 1:0.25 toabout 1:15 (e.g., 1:0.25, 1:0.5, 1:1, 1:2.5, 1:5, 1:10, or 1:15). Forophthalmic use, provided pharmaceutical compositions may be formulatedas micronized suspensions or in an ointment such as petrolatum.

Pharmaceutical compositions of this invention may also be administeredby nasal aerosol or inhalation.

In some embodiments, pharmaceutical compositions of this invention areformulated for intra-peritoneal administration.

The amount of the crystalline form of a compound represented by the bisHCl salt Structural Formula 1 in pharmaceutical compositions of thisinvention is such that is effective to measurably treat or prevent atetracycline-responsive disease or disorder, in a biological sample orin a subject. In certain embodiments, a pharmaceutical composition ofthis invention is formulated for administration to a subject in need ofsuch pharmaceutical composition. The term “subject,” as used herein,means an animal. In some embodiments, the animal is a mammal. In certainembodiments, the subject is a veterinary patient (i.e., a non-humanmammal patient, such as a dog, a cat, a horse, a pig or a rodent, suchas a mouse or rat). In some embodiments, the subject is a dog. In otherembodiments, the subject is a human (e.g., a human patient).

The amount of the crystalline form of a compound represented by the bisHCl salt Structural Formula 1 that may be combined with thepharmaceutically acceptable carrier materials to produce apharmaceutical composition in a single dosage form will vary dependingupon the host treated and/or the particular mode of administration. Inone embodiment, the pharmaceutical compositions should be formulated sothat a dosage of between 0.01-100 mg/kg body weight/day of the compoundof Structural Formula 1 can be administered to a patient receiving thesecompositions. In another embodiment, the dosage is from about 0.5 toabout 100 mg/kg of body weight, or between 1 mg and 1000 mg/dose, every4 to 120 hours, or according to the requirements of the particular drug.Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to about 6 times per day. Exemplary doses,include but are not limited to, 1.0 mg/kg twice a day for about 4-14days and 1.5 mg/kg once a day for 5 to 10 days.

It should also be understood that a specific dosage and treatmentregimen for any particular subject (e.g., patient) will depend upon avariety of factors, including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, and the judgment ofthe treating physician and the severity of the particular disease beingtreated.

Upon improvement of a subject's condition, a maintenance dose of apharmaceutical composition of this invention may be administered, ifnecessary. Subsequently, the dosage or frequency of administration, orboth, may be reduced, as a function of the symptoms, to a level at whichthe improved condition is retained when the symptoms have beenalleviated to the desired level. Subjects may, however, requireintermittent treatment on a long-term basis upon any recurrence ofdisease symptoms.

Methods of Treatment and Uses for Pharmaceutical Compositions

Pharmaceutical compositions described herein are generally useful fortreatment or prevention of a tetracycline-responsive disease ordisorder. Thus, in certain embodiments, the invention provides a methodfor treating a tetracycline-responsive disease or disorder, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a crystalline form, such as a crystalline form, of the bis HClsalt of a compound of Structural Formula 1, or a pharmaceuticalcomposition described herein. The compound of Structural Formula 1 orcrystalline form thereof, pharmaceutical composition thereof orcombination of the foregoing can also be administered to cells inculture, e.g., in vitro or ex vivo, or to a subject, e.g., in vivo, totreat, prevent, and/or diagnose a variety of disorders, including thosedescribed herein.

The term “treat” or “treating” means to alleviate symptoms, eliminatethe causation of the symptoms, either on a temporary or permanent basis,or to slow the appearance of symptoms of the named disorder orcondition.

The term “therapeutically effective amount” means an amount of the bisHCl salt of a compound of Structural Formula 1 or crystalline formthereof (typically, in a pharmaceutical composition described herein)which is effective in treating or lessening the severity of one or moresymptoms of a disorder or condition.

“Tetracycline-responsive disease or disorder” refers to a disease ordisorder that can be treated, prevented, or otherwise ameliorated by theadministration of a tetracycline compound of the present invention.Tetracycline-responsive disease or disorder includes infections, cancer,inflammatory disorders, autoimmune disease, arteriosclerosis, cornealulceration, emphysema, arthritis, osteoporosis, osteoarthritis, multiplesclerosis, osteosarcoma, osteomyelitis, bronchiectasis, chronicpulmonary obstructive disease, skin and eye diseases, periodontitis,osteoporosis, rheumatoid arthritis, ulcerative colitis, prostatitis,tumor growth and invasion, metastasis, diabetes, diabetic proteinuria,panbronchiolitis; aortic or vascular aneurysms, skin tissue wounds, dryeye, bone, cartilage degradation, malaria, senescence, diabetes,vascular stroke, neurodegenerative disorders, cardiac disease, juvenilediabetes, acute and chronic bronchitis, sinusitis, and respiratoryinfections, including the common cold; Wegener's granulomatosis;neutrophilic dermatoses and other inflammatory diseases such asdermatitis herpetiformis, leukocytoclastic vasculitis, bullous lupuserythematosus, pustular psoriasis, erythema elevatum diutinum; vitiligo;discoid lupus erythematosus; pyoderma gangrenosum; pustular psoriasis;blepharitis, or meibomianitis; Alzheimer's disease; degenerativemaculopathy; acute and chronic gastroenteritis and colitis; acute andchronic cystitis and urethritis; acute and chronic dermatitis; acute andchronic conjunctivitis; acute and chronic serositis; uremicpericarditis; acute and chronic cholecystis; cystic fibrosis, acute andchronic vaginitis; acute and chronic uveitis; drug reactions; insectbites; burns and sunburn, bone mass disorder, acute lung injury, chroniclung disorders, ischemia, stroke or ischemic stroke, skin wound, aorticor vascular aneurysm, diabetic retinopathy, hemorrhagic stroke,angiogenesis, and other states for which tetracycline compounds havebeen found to be active (see, for example, U.S. Pat. Nos. 5,789,395;5,834,450; 6,277,061 and 5,532,227, each of which is expresslyincorporated herein by reference).

In addition, the invention relates to methods of treating any disease ordisease state that could benefit from modulating the expression and/orfunction of nitric oxide, metalloproteases, proinflammatory mediatorsand cytokines, reactive oxygen species, components of the immuneresponse, including chemotaxis, lymphocyte transformation, delayedhypersensitivity, antibody production, phagocytosis, and oxidativemetabolism of phagocytes. A method to treat any disease or disease statethat could benefit from modulating the expression and/or function ofC-reactive protein, signaling pathways (e.g., FAK signaling pathway),and/or augment the expression of COX-2 and PGE2 production is covered. Amethod to treat any disease or disease state that could benefit frominhibition of neovascularization is covered.

In certain embodiments, compositions of the invention can be used toprevent or treat important mammalian and veterinary diseases such asdiarrhea, urinary tract infections, infections of skin and skinstructure, ear, nose and throat infections, wound infection, mastitisand the like. In addition, methods for treating neoplasms usingtetracycline compounds of the invention are also included (van derBozert et al., Cancer Res., 48: 6686-6690 (1988)).

Infections that can be treated using compositions of the inventioninclude, but are not limited to, skin infections, GI infections, urinarytract infections (e.g., complication UTI), complicated intra-abdominalinfections, genito-urinary infections, respiratory tract infections,sinuses infections, middle ear infections, systemic infections, cholera,influenza, bronchitis, acne, malaria, sexually transmitted diseaseincluding syphilis and gonorrhea, Legionnaires' disease, Lyme disease,Rocky Mountain spotted fever, Q fever, typhus, bubonic plague, gasgangrene, hospital acquired infections, leptospirosis, whooping cough,anthrax and infections caused by the agents responsible forlymphogranuloma venereum, inclusion conjunctivitis, or psittacosis.Infections can be bacterial, fungal, parasitic and viral infections(including those which are resistant to other tetracycline compounds).

In one embodiment, the infection can be caused by bacteria. In anotherembodiment, the infection is caused by a Gram-positive bacteria. In aspecific aspect of this embodiment, the infection is caused by aGram-positive bacterium selected from Staphylococcus spp., Streptococcusspp., Propionibacterium spp., Enterococcus spp., Bacillus spp.,Corynebacterium spp., Nocardia spp., Clostridium spp., Actinobacteriaspp., and Listeria spp.

In another embodiment, the infection is caused by a Gram-negativebacterium. In one aspect of this embodiment, the infection is caused bya proteobacteria (e.g., Betaproteobacteria and Gammaproteobacteria),including Escherichia coli, Salmonella, Shigella, otherEnterobacteriaceae, Pseudomonas, Moraxella, Helicobacter,Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella oralpha-proteobacteria such as Wolbachia. In another aspect, the infectionis caused by a Gram-negative bacteria selected from cyanobacteria,spirochaetes, green sulfur or green non-sulfur bacteria. In a specificaspect of this embodiment, the infection is caused by a Gram-negativebacteria selected from Enterobactericeae (e.g., E. coli, Klebsiellapneumonia including those containing extended-spectrum .beta.-lactamasesand/or carbapenemases), Bacteroidaceae (e.g., Bacteroides fragilis),Vibrionaceae (Vibrio cholerae), Pasteurellae (e.g., Haemophilusinfluenza), Pseudomonadaceae (e.g., Pseudomonas aeruginosa),Neisseriaceae (e.g. Neisseria meningitidis), Rickettsiae, Moraxellaceae(e.g., Moraxella catarrhalis), any species of Proteeae, Acinetobacterspp., Helicobacter spp., and Campylobacter spp.

In a particular embodiment, the infection is caused by Gram-negativebacterium selected from the group consisting of Enterobactericeae (e.g.,E. coli, Klebsiella pneumoniae), Pseudomonas, and Acinetobacter spp.

In another embodiment, the infection is caused by an organism selectedfrom the group consisting of K. pneumoniae, Salmonella, E. hirae, A.baumanii, M. catarrhalis, H. influenzae, P. aeruginosa, E. faecium, E.coli, S. aureus, and E. faecalis.

In another embodiment, the infection is caused by an organism selectedfrom the group consisting of rickettsiae, chlamydiae, Legionella spp.and Mycoplasma spp.

In another embodiment, the infection is caused by an organism resistantto tetracycline or any member of first and second generation oftetracycline antibiotics (e.g., doxycycline or minocycline).

In another embodiment, the infection is caused by an organism resistantto methicillin.

In another embodiment, the infection is caused by an organism resistantto vancomycin.

In another embodiment, the infection is caused by an organism resistantto a quinolone or fluoroquinolone.

In another embodiment, the infection is caused by an organism resistantto tigecycline.

In another embodiment, the infection is caused by a multidrug-resistantpathogen (having intermediate or full resistance to any two or moreantibiotics). In another embodiment the infection is a Bacillusanthracis infection. “Bacillus anthracis infection” includes any state,diseases, or disorders caused or which result from exposure or allegedexposure to Bacillus anthracis or another member of the Bacillus cereusgroup of bacteria. In another embodiment, the infection is caused byBacillus anthracis (anthrax), Yersinia pestis (plague), or Francisellatularensis (tularemia).

In yet another embodiment, the infection can be caused by more than oneorganism described above. Examples of such infections include, but arenot limited to, intra-abdominal infections (often a mixture of agram-negative species like E. coli and an anaerobe like B. fragilis),diabetic foot (various combinations of Streptococcus, Serratia,Staphylococcus and Enterococcus spp., anaerobes (S. E. Dowd, et al.,PloS one 2008; 3:e3326) and respiratory disease (especially in patientsthat have chronic infections like cystic fibrosis—e.g., S. aureus plusP. aeruginosa or H influenza, atypical pathogens), wounds and abscesses(various gram-negative and gram-positive bacteria, notably MSSA/MRSA,coagulase-negative staphylococci, enterococci, Acinetobacter, P.aeruginosa, E. coli, B. fragilis), and bloodstream infections (13% werepolymicrobial (H. Wisplinghoff, et al., Clin. Infect. Dis. 2004;39:311-317)).

In a further embodiment, the tetracycline responsive disease or disorderis not a bacterial infection. In another embodiment, the compositions ofthe invention are essentially non-antibacterial. For example,non-antibacterial compositions may have MIC values greater than about 4μg/mL. In another embodiment, the compositions of the invention haveboth antibacterial and non-antibacterial effects.

Tetracycline responsive disease or disorder also includes diseases ordisorders associated with inflammatory process associated states (IPAS).The term “inflammatory process associated state” includes states inwhich inflammation or inflammatory factors (e.g., matrixmetalloproteinases (MMPs), nitric oxide (NO), TNF, interleukins, plasmaproteins, cellular defense systems, cytokines, lipid metabolites,proteases, toxic radicals, adhesion molecules, etc.) are involved or arepresent in an area in aberrant amounts, e.g., in amounts which may beadvantageous to alter, e.g., to benefit the subject. The inflammatoryprocess is the response of living tissue to damage. The cause ofinflammation may be due to physical damage, chemical substances,micro-organisms, tissue necrosis, cancer or other agents. Acuteinflammation is short-lasting, lasting only a few days. If it is longerlasting however, then it may be referred to as chronic inflammation.

IPASs include inflammatory disorders. Inflammatory disorders aregenerally characterized by heat, redness, swelling, pain and loss offunction. Examples of causes of inflammatory disorders include, but arenot limited to, microbial infections (e.g., bacterial and fungalinfections), physical agents (e.g., burns, radiation, and trauma),chemical agents (e.g., toxins and caustic substances), tissue necrosisand various types of immunologic reactions.

Examples of inflammatory disorders can be treated using the compounds ofthe invention or a pharmaceutically acceptable salt thereof include, butare not limited to, osteoarthritis, rheumatoid arthritis, acute andchronic infections (bacterial and fungal, including diphtheria andpertussis); acute and chronic bronchitis, sinusitis, and upperrespiratory infections, including the common cold; acute and chronicgastroenteritis and colitis; inflammatory bowel disorder; acute andchronic cystitis and urethritis; vasculitis; sepsis; nephritis;pancreatitis; hepatitis; lupus; inflammatory skin disorders including,for example, eczema, dermatitis, psoriasis, pyoderma gangrenosum, acnerosacea, and acute and chronic dermatitis; acute and chronicconjunctivitis; acute and chronic serositis (pericarditis, peritonitis,synovitis, pleuritis and tendinitis); uremic pericarditis; acute andchronic cholecystis; acute and chronic vaginitis; acute and chronicuveitis; drug reactions; insect bites; burns (thermal, chemical, andelectrical); and sunburn.

IPASs also include matrix metalloproteinase associated states (MMPAS).MMPAS include states characterized by aberrant amounts of MMPs or MMPactivity.

Examples of matrix metalloproteinase associated states (“MMPASs”) can betreated using compositions of the invention include, but are not limitedto, arteriosclerosis, corneal ulceration, emphysema, osteoarthritis,multiple sclerosis (Liedtke et al., Ann. Neurol. 1998, 44: 35-46;Chandler et al., J. Neuroimmunol. 1997, 72: 155-71), osteosarcoma,osteomyelitis, bronchiectasis, chronic pulmonary obstructive disease,skin and eye diseases, periodontitis, osteoporosis, rheumatoidarthritis, ulcerative colitis, inflammatory disorders, tumor growth andinvasion (Stetler-Stevenson et al., Annu. Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys. Acta 1987, 907: 191-217; Liet al., Mol. Carcillog. 1998, 22: 84-89)), metastasis, acute lunginjury, stroke, ischemia, diabetes, aortic or vascular aneurysms, skintissue wounds, dry eye, bone and cartilage degradation (Greenwald etal., Bone 1998,22: 33-38; Ryan et al., Curr. Op. Rheumatol. 1996, 8:238-247). Other MMPAS include those described in U.S. Pat. Nos.5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412; 4,704,383,4,666,897, and RE 34,656, incorporated herein by reference in theirentirety.

In a further embodiment, the IPAS includes disorders described in U.S.Pat. Nos. 5,929,055; and 5,532,227, incorporated herein by reference intheir entirety.

Tetracycline responsive disease or disorder also includes diseases ordisorders associated with NO associated states. The term “NO associatedstates” includes states which involve or are associated with nitricoxide (NO) or inducible nitric oxide synthase (iNOS). NO associatedstate includes states which are characterized by aberrant amounts of NOand/or iNOS. Preferably, the NO associated state can be treated byadministering tetracycline compounds of the invention. The disorders,diseases and states described in U.S. Pat. Nos. 6,231,894; 6,015,804;5,919,774; and 5,789,395 are also included as NO associated states. Theentire contents of each of these patents are hereby incorporated hereinby reference.

Examples of diseases or disorders associated with NO associated statescan be treated using the compositions of the invention include, but arenot limited to, malaria, senescence, diabetes, vascular stroke,neurodegenerative disorders (Alzheimer's disease and Huntington'sdisease), cardiac disease (reperfusion-associated injury followinginfarction), juvenile diabetes, inflammatory disorders, osteoarthritis,rheumatoid arthritis, acute, recurrent and chronic infections(bacterial, viral and fungal); acute and chronic bronchitis, sinusitis,and respiratory infections, including the common cold; acute and chronicgastroenteritis and colitis; acute and chronic cystitis and urethritis;acute and chronic dermatitis; acute and chronic conjunctivitis; acuteand chronic serositis (pericarditis, peritonitis, synovitis, pleuritisand tendonitis); uremic pericarditis; acute and chronic cholecystis;cystic fibrosis, acute and chronic vaginitis; acute and chronic uveitis;drug reactions; insect bites; burns (thermal, chemical, and electrical);and sunburn.

In another embodiment, the tetracycline responsive disease or disorderis cancer. Examples of cancers that can be treated using the compoundsof the invention or a pharmaceutically acceptable salt thereof includeall solid tumors, i.e., carcinomas e.g., adenocarcinomas, and sarcomas.Adenocarcinomas are carcinomas derived from glandular tissue or in whichthe tumor cells form recognizable glandular structures. Sarcomas broadlyinclude tumors whose cells are embedded in a fibrillar or homogeneoussubstance like embryonic connective tissue. Examples of carcinomas whichmay be treated using the methods of the invention include, but are notlimited to, carcinomas of the prostate, breast, ovary, testis, lung,colon, and breast. The methods of the invention are not limited to thetreatment of these tumor types, but extend to any solid tumor derivedfrom any organ system. Examples of treatable cancers include, but arenot limited to, colon cancer, bladder cancer, breast cancer, melanoma,ovarian carcinoma, prostate carcinoma, lung cancer, and a variety ofother cancers as well. The methods of the invention also cause theinhibition of cancer growth in adenocarcinomas, such as, for example,those of the prostate, breast, kidney, ovary, testes, and colon. In oneembodiment, the cancers treated by methods of the invention includethose described in U.S. Pat. Nos. 6,100,248; 5,843,925; 5,837,696; or5,668,122, incorporated herein by reference in their entirety.

Alternatively, the compositions may be useful for preventing or reducingthe likelihood of cancer recurrence, for example, to treat residualcancer following surgical resection or radiation therapy. Thecompositions useful according to the invention are especiallyadvantageous as they are substantially non-toxic compared to othercancer treatments.

In a further embodiment, the compositions of the invention areadministered in combination with standard cancer therapy, such as, butnot limited to, chemotherapy.

Examples of tetracycline responsive states can be treated using thecompositions of the invention or a pharmaceutically acceptable saltthereof also include neurological disorders which include bothneuropsychiatric and neurodegenerative disorders, but are not limitedto, such as Alzheimer's disease, dementias related to Alzheimer'sdisease (such as Pick's disease), Parkinson's and other Lewy diffusebody diseases, senile dementia, Huntington's disease, Gilles de laTourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis(ALS), progressive supranuclear palsy, epilepsy, and Creutzfeldt-Jakobdisease; autonomic function disorders such as hypertension and sleepdisorders, and neuropsychiatric disorders, such as depression,schizophrenia, schizoaffective disorder, Korsakoffs psychosis, mania,anxiety disorders, or phobic disorders; learning or memory disorders,e.g., amnesia or age-related memory loss, attention deficit disorder,dysthymic disorder, major depressive disorder, mania,obsessive-compulsive disorder, psychoactive substance use disorders,anxiety, phobias, panic disorder, as well as bipolar affective disorder,e.g., severe bipolar affective (mood) disorder (BP-1), bipolar affectiveneurological disorders, e.g., migraine and obesity.

Further neurological disorders include, for example, those listed in theAmerican Psychiatric Association's Diagnostic and Statistical manual ofMental Disorders (DSM), the most current version of which isincorporated herein by reference in its entirety.

In another embodiment, the tetracycline responsive disease or disorderis diabetes. Diabetes that can be treated using the compositions of theinvention include, but are not limited to, juvenile diabetes, diabetesmellitus, diabetes type I, or diabetes type II. In a further embodiment,protein glycosylation is not affected by the administration of thecompositions of the invention. In another embodiment, the composition ofthe invention is administered in combination with standard diabetictherapies, such as, but not limited to insulin therapy.

In another embodiment, the tetracycline responsive disease or disorderis a bone mass disorder. Bone mass disorders that can be treated usingthe compounds of the invention or a pharmaceutically acceptable saltthereof include disorders where a subjects bones are disorders andstates where the formation, repair or remodeling of bone isadvantageous. For examples bone mass disorders include osteoporosis(e.g., a decrease in bone strength and density), bone fractures, boneformation associated with surgical procedures (e.g., facialreconstruction), osteogenesis imperfecta (brittle bone disease),hypophosphatasia, Paget's disease, fibrous dysplasia, osteopetrosis,myeloma bone disease, and the depletion of calcium in bone, such as thatwhich is related to primary hyperparathyroidism. Bone mass disordersinclude all states in which the formation, repair or remodeling of boneis advantageous to the subject as well as all other disorders associatedwith the bones or skeletal system of a subject which can be treated withthe compositions of the invention. In a further embodiment, the bonemass disorders include those described in U.S. Pat. Nos. 5,459,135;5,231,017; 5,998,390; 5,770,588; RE 34,656; 5,308,839; 4,925,833;3,304,227; and 4,666,897, each of which is hereby incorporated herein byreference in its entirety.

In another embodiment, the tetracycline responsive disease or disorderis acute lung injury. Acute lung injuries that can be treated using thecompounds of the invention or a pharmaceutically acceptable salt thereofinclude adult respiratory distress syndrome (ARDS), post-pump syndrome(PPS), and trauma. Trauma includes any injury to living tissue caused byan extrinsic agent or event. Examples of trauma include, but are notlimited to, crush injuries, contact with a hard surface, or cutting orother damage to the lungs.

The tetracycline responsive disease or disorders of the invention alsoinclude chronic lung disorders. Examples of chronic lung disorders thatcan be treated using the compounds of the invention or apharmaceutically acceptable salt thereof include, but are not limited,to asthma, cystic fibrosis, chronic obstructive pulmonary disease(COPD), and emphysema. In a further embodiment, the acute and/or chroniclung disorders that can be treated using the compositions of theinvention include those described in U.S. Pat. Nos. 5,977,091;6,043,231; 5,523,297; and 5,773,430, each of which is herebyincorporated herein by reference in its entirety.

In yet another embodiment, the tetracycline responsive disease ordisorder is ischemia, stroke, or ischemic stroke.

In a further embodiment, the compositions of the invention can be usedto treat such disorders as described above and in U.S. Pat. Nos.6,231,894; 5,773,430; 5,919,775 and 5,789,395, incorporated herein byreference.

In another embodiment, the tetracycline responsive disease or disorderis a skin wound. The invention also provides a method for improving thehealing response of the epithelialized tissue (e.g., skin, mucosae) toacute traumatic injury (e.g., cut, burn, scrape, etc.). The methodincludes using a composition of the invention to improve the capacity ofthe epithelialized tissue to heal acute wounds. The method may increasethe rate of collagen accumulation of the healing tissue. The method mayalso decrease the proteolytic activity in the epithelialized tissue bydecreasing the collagenolytic and/or gellatinolytic activity of MMPs. Ina further embodiment, the tetracycline compound of the invention or apharmaceutically acceptable salt thereof is administered to the surfaceof the skin (e.g., topically). In a further embodiment, the compositionsof the invention are used to treat a skin wound, and other suchdisorders as described in, for example, U.S. Pat. Nos. 5,827,840;4,704,383; 4,935,412; 5,258,371; 5,308,839, 5,459,135; 5,532,227; and6,015,804; each of which is incorporated herein by reference in itsentirety.

In yet another embodiment, the tetracycline responsive disease ordisorder is an aortic or vascular aneurysm in vascular tissue of asubject (e.g., a subject having or at risk of having an aortic orvascular aneurysm, etc.). The compositions may be effective to reducethe size of the vascular aneurysm or it may be administered to thesubject prior to the onset of the vascular aneurysm such that theaneurysm is prevented. In one embodiment, the vascular tissue is anartery, e.g., the aorta, e.g., the abdominal aorta. In a furtherembodiment, the compositions of the invention are used to treatdisorders described in U.S. Pat. Nos. 6,043,225 and 5,834,449,incorporated herein by reference in their entirety.

In still another embodiment, the compounds, compositons andpharmaceutical compositins of the invention can be used to treat oralmucositis. In a particular aspect, the oral mucositis is a result ofchemotherapy, radiation or both. In another particular aspect, thesubject having oral mucositis is undergoing chemotherapy and/orradiation therapy for head and neck cancer. In a specific aspect, thehead and neck cancer is selected from: laryngeal; hypopharyngeal; nasalcavity; paranasal sinus; nasopharyngeal; oral; oropharyngeal; andsalivary gland.

Combination Therapies

In some embodiments, a crystalline form of a compound represented by thebis HCl salt Structural Formula 1 (e.g., Form A, B, I or J) isadministered together with an additional “second” therapeutic agent ortreatment. The choice of second therapeutic agent may be made from anyagent that is typically used in a monotherapy to treat the indicateddisease or condition. As used herein, the term “administered together”and related terms refers to the simultaneous or sequentialadministration of therapeutic agents in accordance with this invention.For example, the compound of Structural Formula 1 may be administeredwith another therapeutic agent simultaneously or sequentially inseparate unit dosage forms or together in a unit dosage form.Accordingly, the invention provides a unit dosage form comprising acrystalline form of a compound represented by the bis HCl salt ofStructural Formula 1 (e.g., Form A, B, I or J), an additionaltherapeutic agent, and a pharmaceutically acceptable carrier.

In one embodiment, where a second therapeutic agent is administered to asubject, the effective amount of the crystalline form of a compoundrepresented by the bis HCl salt of Structural Formula 1 is less than itseffective amount would be where the second therapeutic agent is notadministered. In another embodiment, the effective amount of the secondtherapeutic agent is less than its effective amount would be were thecrystalline form of a compound represented by the bis HCl salt ofStructural Formula 1 not administered. In this way, undesired sideeffects associated with high doses of either agent may be minimized.Other potential advantages (including without limitation improved dosingregimens and/or reduced drug cost) will be apparent to those of skill inthe art.

Synthetic Methods

Also provided herein are synthetic methods for preparing crystallineforms of a compound represented by the bis HCl salt of StructuralFormula 1. In some aspects, a mixture of crystalline forms is produced.For example, the mixture may comprise two or more crystalline formsselected from Form A, Form B, Form I, or Form J, more specifically, twoor more crystalline forms selected from Form B, Form I, or Form J. Insome aspects, the mixture does not comprise Form A.

FORM B:

In one embodiment, the invention relates to a method of preparing acrystalline form of the bis-HCl salt of a compound represented byStructural Formula 1:

-   -   wherein the crystalline form is Form B and is characterized by        at least three x-ray powder diffraction peaks at 2θ angles        selected from 9.19°, 9.66°, 23.32°, and 24.35°,    -   the method comprising:        -   (a) adding together the free base, amorphous form of a            compound of Structural Formula 1 and a solvent mixture            comprising methanol, a first portion of ethanol, water and            concentrated HCl, thereby forming a recrystallization            mixture of amorphous compound;        -   (b) adding a second portion of ethanol to the            recrystallization mixture with stirring at a sufficient            amount, for a sufficient time and at a sufficient            temperature to form particles of crystalline Form B; and        -   (c) isolating the particles of crystalline Form B,    -   thereby preparing a composition comprising particles of        crystalline Form B of the bis HCl salt of the compound of        Structural Formula 1.

In certain aspects of the method of preparing Form B, the ratio byvolume of methanol, first portion of ethanol, water and concentrated HClin the recrystallization mixture is from about 1 to about 8 (methanol),from about 1 to about 3 (ethanol), from about 0.1 to about 1.0 (water)and from about 0.1 to about 2 (concentrated HCl) by volume. In aspecific aspect, the volume of the second portion of ethanol is aboutfrom about 4 to about 6 times the volume of the first portion ofethanol.

In another aspect of the method of preparing Form B, stirring of therecrystallization mixture (both with and without the addition of thesecond portion of ethanol) is conducted at a temperature from about 15°C. to about 25° C.

In another aspect of the method of preparing Form B, the particles areisolated by filtration.

In yet another aspect, the method of preparing Form B further comprisesstep washing the particles of crystalline Form B, for example, withethanol or the mixture of methanol, water, and concentrated HCl.

Form I:

In certain embodiments, the invention relates to a method of preparing acrystalline form of the bis-HCl salt of a compound represented byStructural Formula 1:

-   -   wherein the crystalline form is Form I and is characterized by        at least three x-ray powder diffraction peaks at 2θ angles        selected from 7.22°, 7.80°, 10.41°, and 11.11°,    -   the method comprising:        -   (a) suspending crystalline Form B of the compound of            Structural Formula 1 in a mixture of ethanol, water, and            concentrated HCl, thereby forming a slurry of Form B;        -   (b) stirring the slurry of Form B for a sufficient time and            at a sufficient temperature to form a slurry of crystalline            Form I particles; and        -   (c) isolating the particles of crystalline Form I.

In certain aspects of the method of preparing Form I, the ratio ofethanol to water to concentrated HCl by volume in the mixture ofethanol, water, and concentrated HCl is about 4.0-6.0 to about 0.3-0.5to about 0.3-0.5, for example, about 5.0 to about 0.4 to about 0.4, orabout 5.0 to about 0.4 to about 0.35.

In other aspects, the method of preparing Form I further comprises step(b′) adding to the slurry of Form B an amount of the crystalline Form I,thereby forming a seeded mixture.

In another aspect of the method of preparing Form I, stirring the slurryof Form B or the seeded mixture is conducted at a temperature from about18° C. to about 25° C. for about 12 h to about 40 h.

In certain aspects, the method of preparing Form I further comprisesstep (b″) adding to the slurry of Form I an anti-solvent, therebyforming an anti-solvent slurry. For example, the anti-solvent can beethanol. In a particular aspect, the anti-solvent is ethanol, and isadded over about 1 hour to about 15 hours (such as about 1 hour to about5 hours), for example, over about 1 hour, 3 hours or 10 hours.

In another aspect of the method of preparing Form I, the particles areisolated by filtration.

In certain aspects, the method of preparing Form I further comprisesstep (c′) washing the solid particles, for example, with ethanol, or themixture of ethanol, water, and concentrated HCl.

In certain embodiments, the invention relates to a method of preparing acrystalline form of the bis-HCl salt of a compound represented byStructural Formula 1:

-   -   wherein the crystalline form is Form J and is characterized by        at least three x-ray powder diffraction peaks at 2θ angles        selected from 7.02°, 7.80°, 22.13°, and 23.22°,    -   the method comprising:        -   (a) exposing crystalline Form I to a temperature from about            20° C. to about 45° C. at a relative humidity of about 70%            to about 90%; and        -   (b) isolating particles of crystalline Form J.

In certain aspects of the method of preparing Form J, crystalline Form Iis exposed to a temperature of about 40° C. and a relative humidity ofabout 75%.

In other aspects of the method of preparing Form J, crystalline Form Iis exposed to a temperature of about 25° C. and a relative humidity fromabout 75% to about 85%.

In certain embodiments, the invention relates to a method of preparing acrystalline form of the bis-HCl salt of a compound represented byStructural Formula 1:

-   -   wherein the crystalline form is Form A and is characterized by        at least three x-ray powder diffraction peaks at 2θ angles        selected from 3.31°, 6.01°, 6.33°, and 8.73°, the method        comprising:        -   (a) combining together with stirring the bis-HCl salt of a            compound of Structural Formula 1 in amorphous form and a            solvent mixture of ethanol and water, thereby forming a            solution of amorphous compound;        -   (b) adding to the solution of amorphous compound            concentrated HCl and methanol and stirring, thereby forming            a slurry;        -   (c) adding to the slurry an anti-solvent, thereby forming a            slurry of crystalline Form A; and        -   (d) isolating the solid particles of crystalline Form A,    -   thereby preparing a composition comprising particles of        crystalline Form A of the compound of Structural Formula 1.

In certain aspects of the method of preparing Form A, the ratio ofethanol to water by volume in the mixture of ethanol and water is about3.0-5.0 to about 0.3-0.5, for example, about 4.0 to about 0.4.

In certain aspects of the method of preparing Form A, the ratio ofconcentrated HCl to methanol by volume is about 0.2-0.4 to about0.1-0.4, for example, about 0.3 to about 0.2.

In other aspects, the method of preparing Form A further comprises steps(b′) adding to the slurry of amorphous compound an amount of thecrystalline Form A, thereby forming a seeded mixture; and (b″) stirringthe seeded mixture at a temperature from about 20° C. to about 25° C.for about 1 h to about 24 h.

In certain aspects of the method of preparing Form A, the anti-solventis ethanol. In a more particular aspect, the anti-solvent is added overfrom about 2 h to about 6 h, for example, over about 4 h.

In another aspect, the method of preparing Form A further comprisingstep (c′) cooling the slurry of Form A to about 0° C.

In certain embodiments, the invention relates to any of the methodsdescribed herein (methods to produce crystalline Forms I, B, J or A),further comprising step drying the isolated particles, for example atabout 22° C. to about 30° C. for about 12 h to about 5 days (e.g., fromabout 12 h to about 24 h) at about 26° C. for about 18 h, or undernitrogen gas for about 1 h to about 20 h, then under vacuum conditionsfor about 15 h to about 25 h, or under nitrogen gas for about 1 h, thenunder vacuum conditions for about 20 h, or under nitrogen gas for about10 h, then under vacuum conditions at about 25° C.

“Solvent system,” as used herein, refers to a single solvent or amixture of two or more (typically, two) different solvents. Exemplarysolvents for a solvent system include water and organic solvents suchas, but not limited to, methanol, ethanol, diisopropyl ether,2-propanol, ethyl acetate, and isopropyl acetate.

“Inducing formation,” used herein, includes any conditions that inducethe compound of Structural Formula 1 to crystallize as the specifiedcrystalline form, for example, crystalline Form B or crystalline Form I.Inducing formation includes merely allowing solid particles of thespecified crystalline form to precipitate from a solution or slurry, forexample, without actively performing any step. Inducing formation alsoincludes maturing (e.g., aging, with or without cooling, and/or cycling)a solution comprising a compound of Structural Formula 1 in anappropriate solvent system and/or allowing a solution comprising acompound of Structural Formula 1 in an appropriate solvent system toslowly evaporate, with or without cooling. Inducing formation alsoincludes cooling the compound of Structural Formula 1 or a solutionincluding the compound of Structural Formula 1. Other methods ofinducing formation of a crystalline solid are known in the art andinclude, for example, the use of anti-solvents and vapor diffusion. Inpreferred embodiments, inducing formation comprises cooling the compoundof Structural Formula 1 or a solution or slurry including the compoundof Structural Formula 1 in an appropriate solvent system.

Isolating the solid particles of crystalline Form B, Form I, Form J, orForm A is typically effected by filtration and, optionally, rinsing ofthe filtered solids with a solvent (e.g., a chilled solvent), althoughother means of isolating the solid particles are known in the art. Othermeans of isolating the solid particles of crystalline Form B, Form I,Form J, or Form A include, but are not limited to, distilling liquidaway from the solid particles or otherwise drying the solid particles,for example, by heating, by subjecting to reduced pressure (e.g., invacuo) or any combination of the foregoing.

“Room temperature” and “ambient temperature,” as used herein, means atemperature of from about 16° C. to about 25° C.

“Ambient conditions,” as used herein, refers to room temperature andatmospheric pressure conditions.

Drying crystalline Form B, Form I, Form J, or Form A of the compound ofStructural Formula 1 or a mixture comprising two or more crystallineforms of the compound of Structural Formula 1 can be accomplished, forexample, by distilling any liquid present away from the solidcrystalline form(s), by exposing the solid crystalline form(s) toambient conditions or passing a stream of gas, such as nitrogen gas,over the solid crystalline form(s) (and thereby inducing the evaporationor desolvation of any liquid or entrapped volatile substance), bysubjecting the solid crystalline form(s) to reduced pressure (e.g., invacuo) or any combination of the foregoing. Crystalline Form I, inparticular, can be converted to crystalline Form J by drying underconditions in which ethanol can desolvate from crystalline Form I, forexample, by subjecting crystalline Form I to reduced pressure (e.g., invacuo) or by exposing crystalline Form I to ambient conditions orpassing a stream of gas over crystalline Form I.

It is understood that, quite often, in practice, the steps for preparingcrystalline Form B, Form I, Form J, or Form A according to the methodsdescribed herein entail a combination of heating, maturing and/ordrying.

EXEMPLIFICATION General Materials and Methods

As used herein, TP-434-046 is the bis-HCl salt of the compoundrepresented by structural Formula 1.

XRPD

The data presented in this application contain x-ray diffractionpatterns with labeled peaks and tables with peak lists. The range ofdata collected is instrument dependent. Under most circumstances, peakswithin the range of up to about 30° 20 were selected. Roundingalgorithms were used to round each peak to the nearest 0.1° or 0.01° 20,depending upon the instrument used to collect the data and/or theinherent peak resolution. The location of the peaks along the x-axis (°2θ) in both the figures and the tables were determined using proprietarysoftware (TRIADS™ v2.0) and rounded to one or two significant figuresafter the decimal point based upon the above criteria. Peak positionvariabilities are given to within ±0.2° 2Θ based upon recommendationsoutlined in the USP discussion of variability in x-ray powderdiffraction (United States Pharmacopeia, USP 38-NF 33 through S1, <941>Aug. 1, 2015). For d-space listings, the wavelength used to calculated-spacings was 1.5405929 Å, the Cu—K_(α1) wavelength (Phys. Rev. A56(6)4554-4568 (1997)). Variability associated with d-spacing estimates wascalculated from the USP recommendation, at each d-spacing, and providedin the respective tables.

Per USP guidelines, variable hydrates and solvates may display peakvariances greater than 0.2° 2θ and therefore peak variances of 0.2° 2θare not applicable to these materials.

“Prominent Peaks” are a subset of the entire observed peak list.Prominent peaks are selected from observed peaks by identifyingpreferably non-overlapping, low-angle peaks, with strong intensity.

If multiple diffraction patterns are available, then assessments ofparticle statistics (PS) and/or preferred orientation (PO) are possible.Reproducibility among XRPD patterns from multiple samples analyzed on asingle diffractometer indicates that the particle statistics areadequate. Consistency of relative intensity among XRPD patterns frommultiple diffractometers indicates good orientation statistics.Alternatively, the observed XRPD pattern may be compared with acalculated XRPD pattern based upon a crystal structure, if available.Two-dimensional scattering patterns using area detectors can also beused to evaluate PS/PO. If the effects of both PS and PO are determinedto be negligible, then the XRPD pattern is representative of the powderaverage intensity for the sample and prominent peaks may be identifiedas “Representative Peaks.” In general, the more data collected todetermine Representative Peaks, the more confident one can be of theclassification of those peaks.

“Characteristic peaks,” to the extent they exist, are a subset ofRepresentative Peaks and are used to differentiate one crystallinepolymorph from another crystalline polymorph (polymorphs beingcrystalline forms having the same chemical composition). Characteristicpeaks are determined by evaluating which representative peaks, if any,are present in one crystalline polymorph of a compound against all otherknown crystalline polymorphs of that compound to within ±0.2° 20. Notall crystalline polymorphs of a compound necessarily have at least onecharacteristic peak.

TG

TG analyses for the crystalline Forms A, I and J described herein wereperformed using a TA Instruments 2050 thermogravimetric analyzer.Temperature calibration was performed using nickel and Alumel™. Eachsample was placed in a platinum pan and inserted into the TG furnace.The furnace was heated under a nitrogen purge. The data acquisitionparameters are displayed above each thermogram in the figures. Themethod code on the thermogram is an abbreviation for the start and endtemperature as well as the heating rate; e.g., 00-350-10 means “ambientto 350° C., at 10° C./min.”

TG analyses for crystalline Form B was conducted as a TG-FTIR usingNetzsch Thermo-Microbalance TG 209 with Bruker FT-IR Spectrometer Vector22. Al-crucible (open or with micro hole), N₂ atmosphere, heating rate10° C. min⁻¹

DSC

DSC for the crystalline Forms A, I and J described herein was performedusing a TA Instruments Q2000 differential scanning calorimeter.Temperature calibration was performed using NIST-traceable indium metal.Each sample was placed into an aluminum Tzero crimped (T0C) DSC pan,covered with a lid, and the weight was accurately recorded. A weighedaluminum pan configured as the sample pan was placed on the referenceside of the cell. The data acquisition parameters and pan configurationfor each thermogram are displayed in the Figures. The method code on thethermogram is an abbreviation for the start and end temperature as wellas the heating rate, e.g., 30-250-10 means “from −30° C. to 250° C., at10° C./min.” Other abbreviations: T0C=Tzero crimped pan; HS=lidhermetically sealed; HSLP=lid hermetically sealed and perforated with alaser pinhole; C=lid crimped; NC=lid not crimped.

DSC for crystalline Form B described herein was performed using PerkinElmer DSC 7 with closed Au crucibles, heating rate: 10 or 20° C. min⁻¹,range: −50° C. to 250° C.

Example 1—Form A

Preparation of Form a without Seeding

Amorphous TP-434-046 (bis HCl salt) was obtained according to theprocedures detailed in U.S. Pat. No. 8,906,887, the entire content ofwhich is hereby incorporated by reference. Amorphous TP-434-046 wasstirred in a mixture of HCl in methanol (1.25 M, 1V) and HCl in EtOH(1.25 M, 2V) for about 10 minutes, followed by the addition of ethanol(4V). The suspension was stirred at ambient temperature overnight, andthen cooled to 0° C. Form A was then isolated.

Preparation of Form a with Seeding

Second Generation Process—Form A

The crystallization process was started by dissolving TP-434-046 (thebis HCl salt) in a mixture of methanol/ethanol/water/conc. HCl(1.91V/0.84V/0.19V/0.47V) between 0° to 22° C., followed by polishfiltration and rinsing with ethanol (1.75V). The solution was thenseeded with Form A and stirred at ambient temperature for 2 hours togive a suspension. Additional ethanol (5.4V to 1 part of TP-434-046) wasthen added over a period of 1 hour and the mixture was continuouslystirred for 3 hours. After cooling to 0° C. and stirring at 0° C. for 3hours, the solid was isolated by filtration.

Third Generation Process—Form A

In 3rd generation Form A crystallization process amorphous TP-434-046(bis HCl Salt) was dissolved in a mixture of EtOH/water (3.5V/0.4V),followed by polish filtration and rinsing with a solution ofEtOH/Conc.HCl/MeOH (1.5V/0.3V/0.1V). The solution was then seeded withForm A and stirred overnight. Additional ethanol (4V) was added over aperiod of 4 hours. After stirring at ambient temperature for 1 morehour, the suspension was cooled to 0° C. and stirred at 0° C. for 2hours and then isolated.

Form A is a co-solvate with EtOH and water

The XRPD patterns of Form A is depicted in FIG. 1, and the peaks aretabulated in Table 1.

TABLE 1 °2θ d space (Å) Intensity (%)  3.31 ± 0.20 26.651 ± 1.609  17 5.28 ± 0.20 16.739 ± 0.634  9  6.01 ± 0.20 14.705 ± 0.489  100  6.33 ±0.20 13.954 ± 0.441  45  8.28 ± 0.20 10.669 ± 0.257  5  8.73 ± 0.2010.121 ± 0.231  23  9.19 ± 0.20 9.615 ± 0.209 5  9.50 ± 0.20 9.307 ±0.196 6 10.01 ± 0.20 8.830 ± 0.176 6 10.49 ± 0.20 8.425 ± 0.160 19 11.62± 0.20 7.608 ± 0.130 6 11.88 ± 0.20 7.445 ± 0.125 14 12.32 ± 0.20 7.177± 0.116 4 12.89 ± 0.20 6.863 ± 0.106 8 13.36 ± 0.20 6.624 ± 0.099 513.65 ± 0.20 6.484 ± 0.095 15 14.06 ± 0.20 6.293 ± 0.089 19 14.23 ± 0.206.219 ± 0.087 11 14.79 ± 0.20 5.983 ± 0.080 5 15.22 ± 0.20 5.818 ± 0.07615 15.46 ± 0.20 5.726 ± 0.074 14 16.21 ± 0.20 5.464 ± 0.067 14 16.55 ±0.20 5.353 ± 0.064 18

The TGA trace of one of the batches of Form A is depicted in FIG. 2A.Weight losses of approximately 4.7% from ambient to 125° C. and 2.7%from 125 to 200° C. were observed in the TG thermogram. Weight lossoccurring above 200° C. is likely due to decomposition.

The DSC trace of Form A is depicted in FIG. 2B. The DSC thermogramdisplays a broad endotherm at approximately 120° C. that correlates tothe TG data. There is also an endotherm displayed at approximately 222°C. that may be due to a melt/decomposition.

Example 2—Form B

Preparation of Form B without Seeding

Conversion of TP-434-Frebase to TP-434-046 Form B

Procedure:

To a 5 L dried flask was charged 814 ml anhydrous methanol, followed by358 mL 200 proof anhydrous ethanol. The mixture was cooled to 0 to 2° C.Water (81 mL) was charged followed by 199.7 ml 37% concentrated HCl.Temperature rose from 1.8° C. to 19.0° C. After temperature of thereaction mixture returned to 0° C., TP-434 freebase (426 g, about 0.763moles,) was charged as a solid slowly over 30 minutes with temperaturemaintained below 20° C.

Solid suspension appears after only 4 minutes and microscope shows platelike crystal (Form B) formed. The yellow suspension was stirred for 2hours at 20 to 23° C.

Anhydrous 200 proof ethanol (1.91 L) was added over 3 hours into themixture. The resulted yellow suspension was cooled to 0 to 1° C. over 1hour and the suspension was continuously stirred at 0 to 2° C. for 30minutes.

The solid was collected by filtration through “M” fritted filter funnel.The solid cake was washed with 250 ml anhydrous 200 proof ethanol. Afterthe wash, the cake was continuously dried in the filter funnel bypulling vacuum with nitrogen blanket for 14 hours.

The solid (610 g) was transferred into two trays and dry in high vacuumoven at room temperature (25±5° C.) until achieving constant weight(took about 24 hours) to give a yellow solid (466 g). KF is 6.2%. ¹H-NMRshowed the molar ratio of TP-434-046/MeOH/EtOH was 1/0.97/0.16.

The solid (459 g) was further dried under vacuum with humidity (with abeaker of water in the drying oven, 60-80% humidity, vacuum 0.043 mpa)for one day and then dried under vacuum by oil pump for two days toafford TP-434-046 as light yellow solid (458 g).

HPLC of product showed 98.88% TP-434. Assay of w/w showed the productcontained 75.3% TP-434 Freebase. (Based on the w/w assay of the product,the yield of the crystallization is as least 81% without w/w assaycorrection of the starting material.) The residual methanol was 1.3 wt %and ethanol was not detected by ¹H-NMR. The residue water by KF was11.18%. CHN and chloride analysis showed C, 45.07%; H, 5.80%; N, 7.88%;Cl−, 9.88%. Calc. for C₂₇H₃₃C₁₂F₁N₄O₈+5H₂O: C, 44.94%; H, 6.01%; N,7.76%; Cl−, 9.83; water, 12.5%. The XRPD of product was consistent withForm B.

Preparation of Form B with Seeding

Crude TP-434-046 was dissolved in methanol (6V). The solution was polishfiltered through a 0.45 μm inline filter and rinsed with a solution ofmethanol (1V), water (0.56V) and concentrated HCl (0.16V). The solutionwas seeded with Form B seeds (0.5 wt %) prepared as described above andstirred at 21±2° C. for approximately 4 hours. Polished filteredconcentrated HCl (0.28V) was added slowly with temperature controlled at21±2° C. The mixture was stirred at 21±2° C. for about 16 hours, cooledto 0° C. in about 30 minutes and held at 0° C. for 3 hours. The solidwas collected by filtration, washed with a solution ofMeOH/water/concentrated HCl (2×1V, 0.87/0.07/0.06). The solid (Form B)was dried under both vacuum and nitrogen.

Form B was characterized as a methanolate (with 1 eq of methanol) withplate shaped crystals under microscope and distinctive XRPD.Crystallization of Form B provide high recovery yield and good abilityfor removing impurities. While Form B may not be the most desired formfor pharmaceutical use due to the presence of methanol, the desirablepurity and high yield of crystalline Form B from amorphous TP-434-046can be translated into pure crystalline Form I, Form J or a mixturethereof for pharmaceutical use.

Form B is a co-solvate with MeOH (about 1 eq) and water.

The XRPD pattern of Form B IS depicted in FIG. 3 and the peaks aretabulated in Table 2.

TABLE 2 °2θ d space (Å) Intensity (%)  6.10 ± 0.20 14.467 ± 0.474  36 9.19 ± 0.20 9.612 ± 0.209 68  9.48 ± 0.20 9.325 ± 0.196 40  9.66 ± 0.209.149 ± 0.189 100 11.87 ± 0.20 7.450 ± 0.125 19 12.08 ± 0.20 7.319 ±0.121 24 13.05 ± 0.20 6.781 ± 0.104 30 14.37 ± 0.20 6.159 ± 0.085 1614.53 ± 0.20 6.091 ± 0.083 8 15.05 ± 0.20 5.880 ± 0.078 12 16.21 ± 0.205.462 ± 0.067 5 17.63 ± 0.20 5.027 ± 0.057 53 17.77 ± 0.20 4.989 ± 0.05634 18.47 ± 0.20 4.801 ± 0.052 10 18.94 ± 0.20 4.683 ± 0.049 9 19.02 ±0.20 4.662 ± 0.049 10 19.54 ± 0.20 4.538 ± 0.046 19 19.94 ± 0.20 4.449 ±0.044 52 20.48 ± 0.20 4.333 ± 0.042 36 22.08 ± 0.20 4.022 ± 0.036 622.42 ± 0.20 3.962 ± 0.035 7 23.32 ± 0.20 3.811 ± 0.032 63 23.87 ± 0.203.726 ± 0.031 26 24.35 ± 0.20 3.653 ± 0.030 57

The TGA trace of Form B is depicted in FIG. 4A. The TG-FTIR measurementshowed a release of MeOH and small amounts of water in the range of50-150° C. (12.7 wt-% mass loss). The decomposition of the sample wasdetected above 200° C.

A DSC trace of Form B is shown in FIG. 4B. DSC measurement was carriedout in a closed Au-crucible (closed under nitrogen flow). The sample washeated in the range of −50 to 205° C. showing a melting peak at 128° C.(ΔH_(f)=56.3 J/g) and the beginning of the decomposition of the sample.

Example 3—Discovery of Form I/Form J Preparation of Form I

Crystalline Form B was dissolved in EtOH (5 vol) and water (0.4 vol).The mixture was held at 35° C. for 4 hours then cooled to 30° C. andheld for 2 hours. Additional solvents (0.3 vol of conc HCl and 0.1 volof MeOH) were added and the solution was seeded with 5% crystalline FormA then cooled to room temperature. After stirring overnight at roomtemperature, crystals with a new shape were observed. The mixture wasthen heated to 35° C. for 1 hour and cooled to room temperature. Thecrystals were fully converted to the new form. This initial lot of newform was later found to be a mixture of Form J and Form I. Forms I and Jappear as irregularly shaped hexagons, diamonds, or clusters undermicroscope and has a distinctive XRPD.

Example 4—Form I

Form I is a hemi-ethanolate containing about 0.5 eq (or about 3.5 wt %)of ethanol. The water content of Form I is about 3.4% (determined byKF).

Form I is an orthorhombic unit cell, spacegroup P212121, with unit cellparameters:

a=11.8 Angstromsb=12.8 Åc=39.9 Å

Vol=6024 Å³.

Observed XRPD peaks for Form I are shown in FIG. 5, and listed in Table3.

TABLE 3 °2θ d space (Å) Intensity (%)  4.44 ± 0.20 19.886 ± 0.895  11 7.22 ± 0.20 12.235 ± 0.339  100  7.80 ± 0.20 11.325 ± 0.290  88  8.19 ±0.20 10.787 ± 0.263  32  8.71 ± 0.20 10.144 ± 0.232  11  9.59 ± 0.209.216 ± 0.192 10 10.41 ± 0.20 8.490 ± 0.163 44 11.11 ± 0.20 7.960 ±0.143 56 11.62 ± 0.20 7.608 ± 0.130 12 12.17 ± 0.20 7.266 ± 0.119 2513.08 ± 0.20 6.761 ± 0.103 13 13.52 ± 0.20 6.546 ± 0.096 21 13.78 ± 0.206.420 ± 0.093 19 14.49 ± 0.20 6.108 ± 0.084 9 15.00 ± 0.20 5.900 ± 0.07830 15.33 ± 0.20 5.774 ± 0.075 11 15.70 ± 0.20 5.639 ± 0.071 22 15.86 ±0.20 5.584 ± 0.070 17 16.47 ± 0.20 5.378 ± 0.065 33 17.12 ± 0.20 5.176 ±0.060 17 17.47 ± 0.20 5.071 ± 0.058 10 17.79 ± 0.20 4.982 ± 0.056 1118.79 ± 0.20 4.719 ± 0.050 18 19.29 ± 0.20 4.597 ± 0.047 9 19.96 ± 0.204.444 ± 0.044 22 20.44 ± 0.20 4.342 ± 0.042 35 20.91 ± 0.20 4.244 ±0.040 17

By TGA, Form I exhibits approximately 4.8% weight loss from ambient to120° C., followed by a weight loss of approximately 2.2% from 120 to180° C., indicating loss of volatiles upon heating (FIG. 6A). Thedramatic change in the slope of the TGA thermogram at approximately 218°C. is consistent with decomposition. Once the residual ethanol is lost,Form I can be converted into Form J.

The DSC thermogram displays a broad, weak endotherm at approximately116° C. which is concurrent with the first weight loss step by TGA dueto the loss of volatiles. Another weak endotherm is observed atapproximately 171° C. and likely corresponds to the second, smallerweight loss in TGA. A series of sharp overlapping endothermic andexothermic transitions observed above ˜235° C. are likely due todecomposition (FIG. 6B).

Example 5—Form J (De-Solvated Form of Form I)

Form J was identified from a 1-month stability sample of Form I storedat 40° C./75% RH. Form J can be generated by exposing Form I under 40°C./75% RH or at room temperature with 75-85% RH until almost allresidual ethanol is removed. Form J was determined to be the de-solvatedform of Form I.

Form J is orthorhombic, spacegroup P212121 with unit cell parameters

a=11.8 Åb=13.3 Åc=38.5 Å

Vol=6060 Å³.

Observed peaks for Form J are shown in FIG. 7 and listed in Table 4.

TABLE 4 °2θ d space (Å) Intensity (%)  4.58 ± 0.20 19.287 ± 0.842  14 7.02 ± 0.20 12.588 ± 0.358  93  7.80 ± 0.20 11.325 ± 0.290  100  8.07 ±0.20 10.948 ± 0.271  19  8.76 ± 0.20 10.088 ± 0.230  19  9.19 ± 0.209.619 ± 0.209 8  9.57 ± 0.20 9.236 ± 0.193 12 10.25 ± 0.20 8.619 ± 0.16853 11.00 ± 0.20 8.033 ± 0.146 34 11.35 ± 0.20 7.793 ± 0.137 9 11.85 ±0.20 7.464 ± 0.126 20 12.15 ± 0.20 7.279 ± 0.119 12 13.29 ± 0.20 6.654 ±0.100 33 13.60 ± 0.20 6.506 ± 0.095 34 14.09 ± 0.20 6.280 ± 0.089 1114.98 ± 0.20 5.909 ± 0.078 39 15.27 ± 0.20 5.796 ± 0.075 21 15.45 ± 0.205.732 ± 0.074 16 15.64 ± 0.20 5.660 ± 0.072 8 16.21 ± 0.20 5.465 ± 0.06729 16.39 ± 0.20 5.405 ± 0.066 24 17.04 ± 0.20 5.200 ± 0.061 21 17.59 ±0.20 5.037 ± 0.057 18 17.80 ± 0.20 4.979 ± 0.055 10 18.45 ± 0.20 4.804 ±0.052 7 18.84 ± 0.20 4.706 ± 0.050 12 19.17 ± 0.20 4.626 ± 0.048 1120.10 ± 0.20 4.415 ± 0.043 28 20.63 ± 0.20 4.301 ± 0.041 17 21.53 ± 0.204.125 ± 0.038 23 21.92 ± 0.20 4.051 ± 0.037 32 22.13 ± 0.20 4.013 ±0.036 58 22.52 ± 0.20 3.945 ± 0.035 21 23.22 ± 0.20 3.828 ± 0.033 6123.58 ± 0.20 3.770 ± 0.032 18 24.02 ± 0.20 3.702 ± 0.030 30 24.41 ± 0.203.644 ± 0.029 23 25.28 ± 0.20 3.520 ± 0.027 34 26.08 ± 0.20 3.414 ±0.026 28 26.35 ± 0.20 3.380 ± 0.025 25 26.78 ± 0.20 3.326 ± 0.024 2327.21 ± 0.20 3.275 ± 0.024 13 27.41 ± 0.20 3.251 ± 0.023 12 27.90 ± 0.203.195 ± 0.022 29 28.78 ± 0.20 3.100 ± 0.021 14

By TGA, Form J exhibits approximately 5.6% weight loss from ambient to125° C., indicating loss of volatiles upon heating (FIG. 8A). Thedramatic change in the slope of the TGA thermogram at approximately 223°C. is consistent with decomposition.

The DSC thermogram (FIG. 8B) displays a broad endotherm at approximately118° C. which is concurrent with the weight loss step by TGA due to theloss of volatiles. A weak, sharper endotherm is observed atapproximately 236° C. and may be associated with a melt/decomposition.

Example 6—Relationship Between Form I and Form J

The unit cells of Form I and Form J are sufficiently different to callthem different forms (see above). Form I and J have the same space groupand have similar relative dimensions to likely be isostructural in theirpacking arrangement of the molecules. Form I can be completely convertedto Form J by exposure to humidity and heat (e.g. under 45° C./75% RH orheating to 180° C.). The fact that mixtures of Form I and J are seen inmany samples indicates that Form I and Form J represent differentthermodynamic phases.

Example 7—Advantages of Crystalline Forms

Crystalline Forms A, B, I and J have many advantages over amorpohous.The amorphous material is very hygroscopic and can deliquite easily whenexposed to moisture. The amorphous material is unstable and the rate ofepimerizaton is much greater. See the data in Table 5, Table 6 and Table7 below. The bis HCl salt of Structural Formula 1 contains fewerimpurities when isolated as Forms A, B, I and J than when isolated asamorphous (See Table 8 below). In fact, the exceptional purity of Form Bcan be translated into highly pure crystalline Form I, crystalline FormJ or a mixture of crystalline Forms I and J for pharmaceutical use. Byusing the procedures described herein for converting crystalline Form Binto crystalline Forms I, crystalline Form J or a mixture thereof,crystalline Form I, crystalline Form J or a mixture thereof can beisolated as a composition ready for formulation as a pharmaceuticalcomposition (e.g., acceptable purity, readily dissolvable and/orexhibiting good flow properties).

Among Forms A, I and J, Forms I and J were found to have advantages overForm A as the Active Pharmaceutical Ingredient (API). In general, theyare thermodynamically more stable than other forms in the currentcrystallization systems and tolerate wider process/operation ranges;have higher isolation yields than Form A; are faster to filter andeasier to dry than Form A; are much less hygroscopic between 5-85% RHthan Form A; have higher bulk densities and better flow properties thanForm A. The detailed data are discussed in the following.

Example 8—Epimerization Studies Comparison of Form a, Form B andAmorphous at Room Temperature and 40° C.

The major degradant of the compound of Formula 1 (all forms) is theepimer at the C-4 carbon of the A-Ring of tetracycline core. Epimerincreases from a 4-week stability study at both room temperature and 40°C. of amorphous form, Form A and Form B are summarized in Table 5. Theepimer increases of amorphous form at both room temperature and 40° C.are much higher than those of Form A and B.

TABLE 5 Epimer increases in a 4-week Stability Study of Amorphous Form,Form A and Form B at Room Temperature (RT) and 40° C. Form A Form BAmorphous Form Lot# 2414-44-1 CLC-441-41 SC-452-106 Temperature RT 40°C. RT 40° C. RT 40° C. Epimer 0.31% 2.14% 0.34% 3.65% 0.96% 8.18%increase

Additional Epimerization Studies Comparison of Forms a, B, I and J andAmorphous at 5° C. and Room Temperature

Stability testing was conducted over three months by placing the desiredsample in a vial, capping the vial and then placing parafilm over thecap. The vials were stored at the designated temperature. The sampleswere analyzed by HPLC both at the beginning of testing and at the end oftesting.

In one stability study, amorphous form was compared side-by-side withForm A and Form B. The epimer changes in the 3-month stability samplesat both 5° C. and room temperature (RT) are summarized in Table 6. Theepimer increases in the amorphous sample are 3.42% and 5.47%respectively at 5° C. and RT after 3 months, which are much greater thanthose seen for Forms A and B. These data demonstrated that amorphousform epimerizes much faster and is much less stable than Form A and B.

TABLE 6 Epimer increases in a 3-Month Stability Study of Amorphous Form,Form A and Form B at Room Temperature (RT) and 5° C. Form A Form BAmorphous Form Lot# 2414-44-1 CLC-441-41 SC-452-106 Temperature 5° C. RT5° C. RT 5° C. RT Epimer 0.00% 0.58% 0.03% 0.65% 3.42% 5.47% increase

In another stability study, the stability of Forms A, I and J werecompared. The desired stability samples were packaged in double PE bagswith desiccant between the bags and then sealed in a foil bag. Thestability samples were stored under the designated stabilitytemperature. The samples were analyzed by HPLC at initiation of thetesting and at three months.

The corresponding epimer changes in the 3-month stability samples atboth 5° C. and room temperature (RT) are summarized in Table 7. Form Iand J showed epimer amounts comparable to those seen for Form A.

TABLE 7 Epimer changes in a 3-Month Stability Study of Form A, Form Iand Form J at Room Temperature (RT) and 5° C. Form A Form I Form J Lot#924001N14 DL-509-1 CLC-475-72-3 Temperature 5° C. RT 5° C. RT 5° C. RTEpimer −0.14% 0.29% 0.02% 0.75% −0.04% 0.07% changes

The data in both stability studies demonstrated that crystalline formsA, B, I and J were more stable than amorphous form.

Example 9—Dynamic Vapor Sorption/Desorption (DVS)

Moisture sorption/desorption data were collected on a VTI SGA-100 VaporSorption Analyzer for Forms A, I and J. Sorption and desorption datawere collected over a range from 5% to 95% RH at 10% RH increments undera nitrogen purge.

For Amorphous solid, Dynamic Vapor Sorption (DVS) was measured via a SMS(Surface Measurement Systems) DVS Intrinsic instrument. Sorption anddesorption were collected using the following program: 25% RH-95% RH-0%RH-95% RH in 10% RH increments.

For Form B, Dynamic Vapor Sorption (DVS) was measured via a SMA (SurfaceMeasurement Systems) DVS-1 water vapor sorption analyzer. Sorption anddesorption were collected using the following program: 50% RH-0% RH-96%RH-50% RH in 5% RH/h increments.

The DVS Plots are shown in FIGS. 9-13 (amorphous, Form A, Form B, Form Iand Form J, respectively). The DVS data below showed that amorphousTP-434-046 has the most weight gain when relative humidity increasedfrom 25% RH to 95% RH: amorphous (about 35.5%), Form A (about 19.5%),Form B (about 11%), Form I (about 24.5%), Form J (about 21%). These dataare generated from the absorption curve of the DVS as in most cases theform changed before desorption curve start.

Example 10—Impurity Purging Studies

The impurity purging abilities of slurry to slurry conversion of Form Bto Form I/J as described herein are summarized below in Table 8 andcompared with Forms A and B crystallization processes described herein.Of note, the precipitation to generate the amorphous form does not purgeany of the impurities. Form B provides the necessary reduction in theimpurity M-16 (TP-6773), which is difficult to remove. In addition,other impurities are also removed at the necessary levels by Form Bcrystallization (see, e.g., NMP and TP-630). While Form B may not be themost desirable Form described herein to use as an API because it is amethanolate, the high impurity purging ability is advantageous forconversion to thermodynamically stable Form I, Form J or a mixturethereof.

TABLE 8 Impurity Purging (Reduction of Impurity Level) Form A Form BAmorphous Form crystal- crystal- Slurry Form B Impurity Precipitationlization lization to Form I/J M − 16 No purging  5-8% 25-28%  Increased≈10% (TP-6773) Epimer No purging Up to 40%  70% 40-80%   (TP-498) M − 18No purging  90% 30-60%  25-40%   (TP-5799) TP-034 No purging  90%50-79%  50-80%   M − 2 NA NA  50% 90% (TP-4705) M + 14 NA >95%  62% 85%(TP-363) TP-630 54% >95% >99% >99%  NMP 97% >95% >99% >99%  M + 94 NANA >98% 61% (TP-3978)The structures of the impurities are set forth below:

Example 11—Large Scale Batches of Form I and Form J 50-g Scale-Up Batch

A 50-g scale-up of the slurry-to-slurry process was performed. Form Bwas converted to the new form overnight. The supernatant concentrationafter 18 hours was 21 mg/g (TP-434 free base). The solids were filtered(fast filtration), washed with ethanol (1 vol) and dried in vacuum ovenat 26° C. Scheme 1 summarizes the results from this experiment. XRPDpattern of this material confirmed that it was mostly Form I with minorForm J. The freebase (FB) potency of TP-434 was determined by HPLCweight/weight assay using TP-434 reference standard.

100-g Scale-Up Batch

Preparation of Form I was scaled up using 100 g of Form B. Theexperiment was carried out by adding Form B into a solution of 5 vol ofEtOH, 0.4 vol of water, 0.4 of conc. HCl followed by charging Form Iseeds (2.5 wt % seed loading) and a total of 85 g of Form I wasisolated. Scheme 2 summarizes the data. The purity of Form I wasconsistent with previous batches.

300-g Scale-Up Batch

A 300-g scale slurry to slurry conversion from Form B to Form I wasperformed. The batch overview is presented in Scheme 3:

The rate of the 300-g scale crystallization was slower than expected.After overnight stirring at room temperature, the supernatantconcentration for Form I crystallization usually stands at 8-15 mg/mL offree base. For the 300-g scale batch however, this concentration was 39mg/mg. Cooling the suspension to 10° C., then 0° C., did not lower theconcentration as fast as the equivalent operation for Form A. After 30hours overall time, 1 volume of EtOH was added to the suspension at 0°C. The batch was allowed to stir at room temperature up to 48 hourstotal time. At this point, the concentration reached 11 mg/mL and thebatch was filtered, washed and the product dried in vacuum ovenovernight to afford TP-434-046 (Form I)

664-g Scale-Up Batch

Starting with Form B (664 g), the scale-up was carried out using thecurrent Form B to Form I conversion procedure to afford TP-434-046 (575g, 84% yield, containing 3.1% EtOH and 4.5% water) after drying in avacuum oven. The batch overview is shown in Scheme 4. The XRPD wasconsistent with Form I with some Form J.

1.83-kg Scale-Up Batch

A Scale-up batch was made starting with 1.83 kg of Form B. The detailedprocedure is described in the followings.

TABLE 9 Material Qty MW Mole eq. TP-434-046 form B 1830 g 631.5 1.0 EtOH200 proof 9150 mL — 5 V Water-HPLC grade 732 mL — 0.4 V Conc HCl 732 mL— 0.4 V TP-434-046 form I seeds 90 g 631.5 5% wt EtOH 200 proof 5490 mL— 3 V EtOH 200 proof 3294 mL — 1.8 V Water 183 mL — 0.1 V Conc HCl 183mL — 0.1 V

-   -   1. To a 20 L reactor, charge EtOH (9150 mL), H₂O (732 mL) and        concentrated HCl (732 mL). Note: Filtered the solution through        the 0.22-μm filter prior to use.    -   2. Charge TP-434-046 form B (1830 g) in one portion and stir for        5 minutes to allow the solution to reach saturation.    -   3. Seed with form I (90 g) in one portion and stir at room        temperature overnight 18 hours.    -   4. Check the Microscope, form B convert into Form I. Note:        Confirm FORM I    -   5. Supernatant wt/wt assay is 1.77% using HPLC.    -   6. Add ethanol (5490 mL, 2 vol) over 3 hours. Note: Filter the        solution through the 0.22-μm filter prior to use.    -   7. Stir the reaction mixture one more hour at room temperature.    -   8. Filter and collect the solids by 18-inch crock filter.    -   9. Then rinse the cake with 3660 mL of a rinsing solution made        up of the initial solvent ratio (3294 mL EtOH/183 mL H₂O/183 mL        conc HCl). Note: Filter the solution through the 0.22 μm filter        prior to use.    -   10. Dry under nitrogen flow for overnight [EtOH (By HNMR): 3.63        wt %; KF: 6.27%]    -   11. Dry under vacuum oven to get constant weight at 25° C.        -   EtOH (By HNMR): 3.45 wt %        -   KF: 3.83%

Formation of Form I in Pilot Plant

Crude TP-434-046 was crystallized to give Form B, followed byslurry-to-slurry conversion of Form B to Form I/J.

Form B was charged into a mixture of EtOH/water/conc. HCl (5V/0.4V/0.4V)and stirred between 15° C. to 25° C. (targeting 21° C.) for 5 to 15minutes (targeting 10 min) followed by the addition of Form I seeds (5wt %). The mixture was stirred between 18° C. to 23° C. (targeting 21°C.) for 10 to 18 hours (targeting 14 hours). A sample was taken andanalyzed for supernatant concentration and microscope. If thesupernatant concentration was ≤6.0% and exclusive formation of Form I/Jconfirmed by microscope, ethanol (3V) was added over a minimum of 3hours between 18° C. to 23° C. (targeting 21° C.). The mixture was thenstirred for minimum of 1 hour before filtering.

In the first production, after the Form B slurry was seeded with Form Iand stirred for 14 hours at 21° C. with stirring rate of 80 rpm,microscope showed a mixture of Form A and Form I/J. In order to speed upthe conversion of Form A to Form I in production of FP-000205, thetemperature was increased from 21° C. to about 22.5° C. (still withinthe defined temperature range of 18° C. to 23° C.) and the stirringspeed was increased from 80 rpm to 120 rpm. Within 6 hours, all From Awas converted to Form I. The supernatant concentration of TP-434-046 was3.6%, meeting the specification of ≤6.0%. Ethanol (3V) was charged over3 hours and the mixture was stirred for about 10 hours before filtering.

To avoid the formation of Form A and increase the rate of Form A to FormI/J conversion in case Form A does form, some modifications were madefor the next two productions. The aging temperature and duration wereboth increased from between 18° C. to 23° C. (targeting 21° C.) for 10to 18 hours (targeting 14 hours) to between 18° C. to 25° C. for 24 to36 hours (targeting 24 hours); while targeting the upper part of thetemperature range in the first 12 hours and targeting 21° C. in thesecond 12 hours.

The productions of the last two batches were executed according to therevised procedure. After the seeded mixtures were stirred at about 24°C. for about 12 hours, no presence of Form A was detected by microscopein either batch. After continuously stirring for 12 hours at 21° C.,samples were taken and the supernatant concentrations were 2.5% and 2.8%respectively. After anti-solvent (EtOH 3V) addition over 3 hours, themixture in 2nd batch was stirred for about 8 hours and the mixture inthird batch was stirred for about 3 hours before filtration.

The brief summary of results on yield, XRPD, residual solvent, particlesize and HPLC purity are combined in Table 10.

TABLE 10 Summary of Yields, XRPD, Residual Solvents, Particle size andHPLC purity of the Three Pilot Plant Batches HPLC Analysis XRPD EtOH/KFD10/D50/D90 TP-434/M − 16/Epimer/ Batch# Yield Interpretation (IPC data)(μm) M − 2/M − 18/TP-034 1 87.9% Consistent 3.2%/5.8% 28.6/60.7/154.098.14/0.75/0.63/<0.05/ with Form I (3.65%/5.1%) 0.42/0.06 2 80.3%Consistent 3.5%/4.5%  89.8/137.2/211.9 97.97/0.87/0.83/ND/ with Form I(3.43%/3.6%) 0.23/0.10 plus Form J 3 77.5% Consistent 2.8%/4.6%111.8/172.3/264.8 97.79/0.89/0.73/ND/ with Form I (3.18%/5%)   0.38/0.21plus Form J

Based on XRPDs data, Batch 1 contains only Form I, as demonstrated bythe smooth shoulder for the first peak of Form I around 7° Θ. Batch 2 isconsistent with Form I plus minor Form J, as demonstrated by the widenedshoulder of the first peak of Form I around 7° 6. Batch 3 contains moreForm J, as demonstrated by the peak on the shoulder of the first peak ofForm I around 7° Θ. All three registration batches met the XRPD specdefined as “consistent to Form I, Form J, or a mixture of Forms I andJ.”

With respect to Batch 1, which contains pure Form I, the formation ofForm A as transient kinetic form during the Form B to Form I conversionmay have affected the product XRPD and particle size. The particles inBatch 1 are mostly composed of smaller well-defined individual crystals,while the particles in Batch 2 and Batch 3 are composed with largecrystals containing clusters of individual crystals. The morphologies ofcrystals of the three batches were also confirmed by polarized lightmicroscopy on the isolated products (data not shown). The observationsunder microscope were also confirmed by the particle size distributiondata where the D50 of Batch 1 is less than half of what were in Batch 2and Batch 3, and much wilder particle size distribution was observed inBatch 1 (data not shown).

TGA and DSC analyses of the three batches were substantially identical.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-11. (canceled)
 12. A crystalline form of the bis-HCl salt of acompound represented by Structural Formula 1:

wherein the crystalline form is Form J and is characterized by at leastthree x-ray powder diffraction peaks at 2θ angles selected from 7.02°,7.80°, 22.13°, and 23.22°.
 13. The crystalline form of claim 12, whereinthe crystalline form is characterized by at least four x-ray powderdiffraction peaks at 2θ angles selected from 7.02°, 7.80°, 10.25°,22.13°, and 23.22°.
 14. The crystalline form of claim 12, wherein thecrystalline form is characterized by at least five x-ray powderdiffraction peaks at 2θ angles selected from 7.02°, 7.80°, 10.25°,11.00°, 13.29°, 13.60°, 14.98°, 21.92°, 22.13°, 23.22°, 24.02° and25.28°.
 15. The crystalline form of claim 12, wherein the crystallineform is characterized by x-ray powder diffraction peaks at 2θ angles of7.02°, 7.80°, 10.25°, 22.13°, and 23.22°.
 16. The crystalline form ofclaim 12, wherein the crystalline form is characterized by x-ray powderdiffraction peaks at 2θ angles of 7.02°, 7.80°, 10.25°, 11.00°, 13.29°,13.60°, 14.98°, 21.92°, 22.13°, 23.22°, 24.02° and 25.28°.
 17. Thecrystalline form of claim 12, wherein the crystalline form ischaracterized by x-ray powder diffraction peaks at 2θ angles of 7.02°,7.80°, 10.25°, 11.00°, 11.85°, 13.29°, 13.60°, 14.98°, 15.27°, 16.21°,16.39°, 17.04°, 20.10°, 21.53°, 21.92°, 22.13°, 22.52°, 23.22°, 24.02°,24.41°, 25.28°, 26.08°, 26.35°, 26.78°, and 27.90°.
 18. The crystallineform of claim 12, wherein the crystalline form is characterized by anx-ray powder diffraction pattern substantially in accordance with thatdepicted in FIG.
 7. 19-27. (canceled)
 28. A crystalline form of thebis-HCl salt of a compound represented by Structural Formula 1:

wherein the crystalline form is Form B and is characterized by at leastthree x-ray powder diffraction peaks at 2θ angles selected from 9.19°,9.66°, 23.32°, and 24.35°.
 29. The crystalline form of claim 28, whereinthe crystalline form is characterized by at least four x-ray powderdiffraction peaks at 2θ angles selected from 9.19°, 9.66°, 17.63°,23.32°, and 24.35°.
 30. The crystalline form of claim 28, wherein thecrystalline form is characterized by at least five x-ray powderdiffraction peaks at 2θ angles selected from 6.10°, 9.19°, 9.48°, 9.66°,13.05°, 17.63°, 17.77°, 19.94°, 20.48°, 23.32° and 24.35°. 31.(canceled)
 32. The crystalline form of claim 28, wherein the crystallineform is characterized by x-ray powder diffraction peaks at 2θ angles of9.19°, 9.66°, 17.63°, 23.32°, and 24.35°.
 33. The crystalline form ofclaim 28, wherein the crystalline form is characterized by at least fivex-ray powder diffraction peaks at 2θ angles selected from 6.10°, 9.19°,9.48°, 9.66°, 13.05°, 17.63°, 17.77°, 19.94°, 20.48°, 23.32°, 23.87° and24.35°.
 34. The crystalline form of claim 28, wherein the crystallineform is characterized by x-ray powder diffraction peaks at 2θ angles of6.10°, 9.19°, 9.48°, 9.66°, 12.08°, 13.05°, 17.63°, 17.77°, 19.54°,19.94°, 20.48°, 23.32°, 23.87°, and 24.35°.
 35. The crystalline form ofclaim 28, wherein the crystalline form is characterized by an x-raypowder diffraction pattern substantially in accordance with thatdepicted in any of FIG.
 3. 36. The crystalline form of claim 28, whereinthe compound of Structural Formula 1 is in the form of a solvate. 37.The crystalline form of claim 28, wherein the compound of StructuralFormula 1 is in the form of a co-solvate.
 38. The crystalline form ofclaim 37, wherein the solvate is a water and methanol co-solvate. 39-41.(canceled)
 42. A pharmaceutical composition, comprising the crystallineform of claim 12 and a pharmaceutically acceptable carrier.
 43. A methodfor treating or preventing an infection is caused by bacteria, themethod comprising administering to a subject in need thereof atherapeutically or prophylactically effective amount of the crystallineform of the composition of claim
 42. 44-45. (canceled)
 46. The method ofclaim 43, wherein the infection is caused by a Gram-positive bacterium.47. The method of claim 43, wherein the infection is caused by aGram-negative bacterium. 48-52. (canceled)